Cytokinesis requires elaborate processes in the midbody, including the timely inactivation of Aurora B kinase (AurB) and membrane fission to separate the daughter cells. Here, we show that human adenovirus (AdV) perturbs midbody function, prevents daughter separation and induces cytoplasmic regression of both cancer cells and primary diploid human airway basal cells resulting in polyploidy. Infected cells undergo cleavage furrow regression after midbody formation driven by the viral protein E4orf4 independently of E4orf4 interaction with protein phosphatase 2A. The co-activator of the anaphase-promoting complex/cyclosome (APC/C), CDC20 is upregulated in AdV infection and required for regression. E4orf4 directly interacts with CDC20 and redirects the E3-ubiquitin ligase APC/C-CDC20 towards AurB, promoting hyperubiquitination and premature extraction of AurB from the midbody. Loss of midbody-associated AurB coincides with midbody collapse and furrow regression. Our findings reveal a previously unrecognized APC/C-CDC20-dependent pathway controlling late cytokinesis through AurB extraction from the midbody.

Hepatitis E virus (HEV) is the leading cause of viral hepatitis worldwide. We find that the HEV ORF2 capsid protein contains intrinsically disordered regions (IDRs) at its N terminus that promote condensate formation through liquid-liquid phase separation both in vitro and in cellulo. Using correlative light and electron microscopy/tomography of HEV-infected cells, we observe that ORF2 forms higher order assemblies composed of orderly stacked tubular filaments resembling amyloid fibrils. By light microscopy, we detect ORF2 fibrils in multiple HEV-infected cell types, including human induced pluripotent stem cell-derived hepatocytes and neurons. Moreover, we discover that the central adapter protein TANK-binding kinase 1 is sequestered by ORF2 fibrils, thereby enabling immune evasion and enhancing viral replication. Together, our findings uncover a previously unrecognized mechanism by which HEV modulates host defense pathways and place HEV among the growing repertoire of viruses that exploit dynamic phase behavior as a broader strategy to regulate virus-host interactions.

Dengue fever represents a significant medical and socio-economic burden in (sub)tropical regions, yet antivirals for treatment or prophylaxis are lacking. JNJ-A07 was described as highly active against the different genotypes within each serotype of the disease-causing dengue virus (DENV). Based on clustering of resistance mutations it has been assumed to target DENV non-structural protein 4B (NS4B). Using a photoaffinity labeling compound with high structural similarity to JNJ-A07, here we demonstrate binding to NS4B and its precursor NS4A-2K-NS4B. Consistently, we report recruitment of the compound to intracellular sites enriched for these proteins. We further specify the mechanism-of-action of JNJ-A07, which has virtually no effect on viral polyprotein cleavage, but targets the interaction between the NS2B/NS3 protease/helicase complex and the NS4A-2K-NS4B cleavage intermediate. This interaction is functionally linked to de novo formation of vesicle packets (VPs), the sites of DENV RNA replication. JNJ-A07 blocks VPs biogenesis with little effect on established ones. A similar mechanism-of-action was found for another NS4B inhibitor, NITD-688. In summary, we unravel the antiviral mechanism of these NS4B-targeting molecules and show how DENV employs a short-lived cleavage intermediate to carry out an early step of the viral life cycle.

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, the ApoE-LPs transport pathway 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 newly synthesized ApoE-LPs accumulate in CD63-positive endosomes of hepatocytes. In addition, we observed the co-egress of ApoE-LPs and CD63-positive intraluminal vesicles (ILVs), which are precursors of extracellular vesicles (EVs), along the late endosomal trafficking route in a microtubule-dependent manner. A fraction of ApoE-LPs associated with CD63-positive EVs appears to be co-transmitted from cell to cell. Given the important role of ApoE in viral infections, we employed as well-studied model the hepatitis C virus (HCV) and found that the viral replicase component nonstructural protein 5A (NS5A) is enriched in ApoE-containing ILVs. Interaction between NS5A and ApoE is required for the efficient release of ILVs containing HCV 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.

ABSTRACT Positive-strand RNA viruses subvert the cellular endomembrane system for the generation of distinct compartments termed replication organelles (ROs) that harbor the site where viral RNAs are generated. In this study, we corroborate that the SARS-CoV-2 non-structural proteins 3 and 4 (nsp3 and nsp4) suffice to remodel the endoplasmic reticulum to form double-membrane vesicles similar to ROs observed in viral infection. Cellular membrane alterations induced by nsp3/4 expression were evaluated through electron tomography and confocal microscopy, and nsp3/4-associated host factors were identified using mass spectrometry. The role of these host factors in virus infection was determined using gene silencing, identifying several host proteins involved in the SARS-CoV-2 replication cycle. Combining the gene silencing approach with ultrastructural analysis of nsp3/4-expressing cells, we found that the host dependency factors FAM149B1, CCAR2, and ZC3HAV1 play a role in the formation of double-membrane vesicles in a replication-independent manner. IMPORTANCE Remodeling of the cellular endomembrane system by viruses allows for efficient and coordinated replication of the viral genome in distinct subcellular compartments termed replication organelles. As a critical step in the viral life cycle, replication organelle formation is an attractive target for therapeutic intervention, but factors central to this process are only partially understood. In this study, we corroborate that two viral proteins, nsp3 and nsp4, are the major drivers of membrane remodeling in SARS-CoV-2 infection. We further report a number of host cell factors interacting with these viral proteins and supporting the viral replication cycle, some of them by contributing to the formation of the SARS-CoV-2 replication organelle.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) remodels the endoplasmic reticulum (ER) to form replication organelles, leading to ER stress and unfolded protein response (UPR). However, the role of specific UPR pathways in infection remains unclear. Here, we found that SARS-CoV-2 infection causes marginal activation of signaling sensor IRE1α leading to its phosphorylation, clustering in the form of dense ER-membrane rearrangements with embedded membrane openings, and XBP1 splicing. By investigating the factors regulated by IRE1α-XBP1 during SARS-CoV-2 infection, we identified stress-activated kinase NUAK2 as a novel host-dependency factor for SARS-CoV-2, HCoV-229E, and MERS-CoV entry. Reducing NUAK2 abundance or kinase activity impaired SARS-CoV-2 particle binding and internalization by decreasing cell surface levels of viral receptors and viral trafficking likely by modulating the actin cytoskeleton. IRE1α-dependent NUAK2 levels were elevated in SARS-CoV-2-infected and bystander non-infected cells, promoting viral spread by maintaining ACE2 cell surface levels and facilitating virion binding to bystander cells.

For successful infection of host cells and virion production, enveloped viruses, including Zika virus (ZIKV), extensively rely on cellular lipids. However, how virus protein–lipid interactions contribute to the viral life cycle remains unclear. Here, we employ a chemo-proteomics approach with a bifunctional cholesterol probe and show that cholesterol is closely associated with the ZIKV structural protein prM. Bioinformatic analyses, reverse genetics alongside with photoaffinity labeling assays, and atomistic molecular dynamics simulations identified two functional cholesterol binding motifs within the prM transmembrane domain. Loss of prM–cholesterol association has a bipartite effect reducing ZIKV entry and leading to assembly defects. We propose a model in which membrane-resident M facilitates cholesterol-supported lipid exchange during endosomal entry and, together with cholesterol, creates a platform promoting virion assembly. In summary, we identify a bifunctional role of prM in the ZIKV life cycle by mediating viral entry and virus assembly in a cholesterol-dependent manner.

Hepatitis C virus (HCV) is highly diverse and grouped into eight genotypes (gts). Infectious cell culture models are limited to a few subtypes and isolates, 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, likely due to cell-to-cell transmission, which appeared to substantially contribute to spreading of other isolates as well. 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 an 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.

Dengue virus (DENV) constitutes one of the most important arboviral pathogens affecting humans. The high prevalence of DENV infections, which cause more than 20,000 deaths annually, and the lack of effective vaccines or direct-acting antiviral drugs make it a global health concern. DENV genome replication occurs in close association with the host endomembrane system, which is remodeled to form the viral replication organelle that originates from endoplasmic reticulum (ER) membranes. To date, the viral and cellular determinants responsible for the biogenesis of DENV replication organelles are still poorly defined. The viral nonstructural protein 4A (NS4A) can remodel membranes and has been shown to associate with numerous host factors in DENV-replicating cells. In the present study, we used reverse and forward genetic screens and identified sites within NS4A required for DENV replication. We also mapped the determinants in NS4A required for interactions with other viral proteins. Moreover, taking advantage of our recently developed polyprotein expression system, we evaluated the role of NS4A in the formation of DENV replication organelles. Together, we report a detailed map of determinants within NS4A required for RNA replication, interaction with other viral proteins, and replication organelle formation. Our results suggest that NS4A might be an attractive target for antiviral therapy. IMPORTANCE DENV is the most prevalent mosquito-borne virus, causing around 390 million infections each year. There are no approved therapies to treat DENV infection, and the only available vaccine shows limited efficacy. The viral nonstructural proteins have emerged as attractive drug targets due to their pivotal role in RNA replication and establishment of virus-induced membranous compartments, designated replication organelles (ROs). The transmembrane protein NS4A, generated by cleavage of the NS4A-2K-4B precursor, contributes to DENV replication by unknown mechanisms. Here, we report a detailed genetic interaction map of NS4A and identify residues required for RNA replication and interaction between NS4A-2K-4B and NS2B-3 as well as NS1. Importantly, by means of an expression-based system, we demonstrate the essential role of NS4A in RO biogenesis and identify determinants in NS4A required for this process. Our data suggest that NS4A is an attractive target for antiviral therapy.

Zika virus (ZIKV) is a mosquito-borne pathogen classified by the World Health Organization (WHO) as a public health emergency of international concern in 2016, and it is still identified as a priority disease. Although most infected individuals are asymptomatic or show mild symptoms, a risk of neurologic complications is associated with infection in adults. Additionally, infection during pregnancy is directly linked to microcephaly and other congenital malformations. Since there are no currently available vaccines or approved therapeutics for this virus, there is a critical unmet need in developing treatments to prevent future ZIKV outbreaks. Toward this end, we performed a large-scale cell-based high-content screen of 51,520 chemical compounds to identify potential antiviral drug candidates. The compound (2E)-N-benzyl-3-(4-butoxyphenyl)prop-2-enamide (SBI-0090799) was found to inhibit replication of multiple ZIKV strains and in different cell systems. SBI-0090799 did not affect viral entry or RNA translation but suppressed RNA replication by preventing the formation of the membranous replication compartment. Selection of drug-resistant viruses identified single-amino-acid substitutions in the N-terminal region of nonstructural protein NS4A, arguing this is the likely drug target These resistance mutations rescued viral RNA replication and restored the formation of the membranous replication compartment. This mechanism of action is similar to clinically approved NS5A inhibitors for hepatitis C virus (HCV). Taken together, SBI-0090799 represents a promising lead candidate for the development of an antiviral treatment against ZIKV infection for the mitigation of severe complications and potential resurgent outbreaks of the virus. IMPORTANCE This study describes the elucidation of (2E)-N-benzyl-3-(4-butoxyphenyl) prop-2-enamide (SBI-0090799) as a selective and potent inhibitor of Zika virus (ZIKV) replication using a high-throughput screening approach. Mapping and resistance studies, supported by electron microscopy observations, indicate that the small molecule is functioning through inhibition of NS4A-mediated formation of ZIKV replication compartments in the endoplasmic reticulum (ER). Intriguingly, this defines a novel nonenzymatic target and chemical matter for the development of a new class of ZIKV antivirals. Moreover, chemical modulation affecting this nonstructural protein mirrors the identification and development of hepatitis C virus (HCV) NS5A inhibitor daclatasvir and its derivatives, similarly interfering with the formation of the viral replication compartment and also targeting a protein with no enzymatic activity, which have been part of a curative strategy for HCV.

Double membrane vesicles (DMVs) serve as replication organelles of plus-strand RNA viruses such as hepatitis C virus (HCV) and SARS-CoV-2. Viral DMVs are morphologically analogous to DMVs formed during autophagy, but 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 and SARS-CoV-2 replication and proper DMV formation. 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 and their pharmacological inhibition also impaired HCV and SARS-CoV-2 replication as well as formation of autophagosome-like DMVs. These data identify PA as host cell lipid involved in proper 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. Host-targeting therapy aiming at PA synthesis pathways might be suitable to attenuate replication of these viruses.

Positive-strand RNA viruses replicate in close association with rearranged intracellular membranes. For hepatitis C virus (HCV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), these rearrangements comprise endoplasmic reticulum (ER)-derived double membrane vesicles (DMVs) serving as RNA replication sites. Cellular factors involved in DMV biogenesis are poorly defined. Here, we show that despite structural similarity of viral DMVs with autophagosomes, conventional macroautophagy is dispensable for HCV and SARS-CoV-2 replication. However, both viruses exploit factors involved in autophagosome formation, most notably class III phosphatidylinositol 3-kinase (PI3K). As revealed with a biosensor, PI3K is activated in cells infected with either virus to produce phosphatidylinositol 3-phosphate (PI3P) while kinase complex inhibition or depletion profoundly reduces replication and viral DMV formation, The PI3P-binding protein DFCP1, recruited to omegasomes in early steps of autophagosome formation, participates in replication and DMV formation of both viruses. These results indicate that phylogenetically unrelated HCV and SARS-CoV-2 exploit similar components of the autophagy machinery to create their replication organelles.

Dengue virus (DENV) and Zika virus (ZIKV), members of the Flavivirus genus, rearrange endoplasmic reticulum membranes to induce invaginations known as vesicle packets (VPs), which are the assumed sites for viral RNA replication. Mechanistic information on VP biogenesis has so far been difficult to attain due to the necessity of studying their formation under conditions of viral replication, where perturbations reducing replication will inevitably impact VP formation. Here, we report a replication-independent expression system, designated pIRO (plasmid-induced replication organelle formation) that induces bona fide DENV and ZIKV VPs that are morphologically indistinguishable from those in infected cells. Using this system, we demonstrate that sequences in the 3' terminal RNA region of the DENV, but not the ZIKV genome, contribute to VP formation in a non-replicative manner. These results validate the pIRO system that opens avenues for mechanistically dissecting virus replication from membrane reorganization.

Positive-strand RNA viruses replicate in distinct membranous structures called replication organelles (ROs). Mechanistic studies of RO formation have been difficult because perturbations affecting viral replication have an impact on viral protein amounts, thus affecting RO biogenesis. Here, we present a detailed guide on how to use a replication-independent expression system, designated pIRO (plasmid-induced replication organelle formation), inducing bona fide flavivirus ROs in transfected cells. This will be useful for mechanistic studies of viral and cellular factors driving flavivirus RO biogenesis. For complete details on the use and execution of this protocol, please refer to Cerikan et al. (2020).

Positive-strand RNA viruses, such as dengue virus (DENV), induce the extensive rearrangement of intracellular membranes that serve as a scaffold for the assembly of the viral replication machinery. In the case of DENV, the main endomembrane ultrastructure produced in infected cells consists of invaginations of the endoplasmic reticulum, designated vesicle packets (VPs), which are the assumed sites of viral RNA replication. VPs are observed as arrays of vesicles surrounded by an outer membrane, the formation of which is induced by the viral nonstructural proteins, presumably in conjunction with specific host factors. However, little is known about the mechanisms governing VP formation, which is mainly due to the lack of a replication-independent system supporting the biogenesis of these membranous structures. Here we describe an expression-based, viral RNA replication-independent, DENV polyprotein system, designated as pIRO (plasmid-induced replication organelle), which is sufficient to induce VP formation. We show that VPs induced by pIRO expression are morphologically indistinguishable from those found in infected cells, suggesting that DENV replication organelle formation does not require RNA replication. We conclude that the pIRO system is a novel and valuable tool that can be used to dissect the mechanisms underlying DENV replication organelle formation.

The hepatitis C virus (HCV) is a major cause of chronic liver disease, affecting around 71 million people worldwide. Viral RNA replication occurs in a membranous compartment composed of double-membrane vesicles (DMVs), whereas virus particles are thought to form by budding into the endoplasmic reticulum (ER). It is unknown how these steps are orchestrated in space and time. Here, we established an imaging system to visualize HCV structural and replicase proteins in live cells and with high resolution. We determined the conditions for the recruitment of viral proteins to putative assembly sites and studied the dynamics of this event and the underlying ultrastructure. Most notable was the selective recruitment of ER membranes around lipid droplets where structural proteins and the viral replicase colocalize. Moreover, ER membranes wrapping lipid droplets were decorated with double membrane vesicles, providing a topological map of how HCV might coordinate the steps of viral replication and virion assembly.

Viral membrane proteins are prime targets in combatting infection. Still, the determination of their structure remains a challenge, both with respect to sample preparation and the need for structural methods allowing for analysis in a native-like lipid environment. Cell-free protein synthesis and solid-state NMR spectroscopy are promising approaches in this context, the former with respect to its great potential in the native expression of complex proteins, and the latter for the analysis of membrane proteins in lipids. Herein, we show that milligram amounts of the small envelope protein of the duck hepatitis B virus (DHBV) can be produced by cell-free expression, and that the protein self-assembles into subviral particles. Proton-detected 2D NMR spectra recorded at a magic-angle-spinning frequency of 110 kHz on <500 μg protein show a number of isolated peaks with line widths comparable to those of model membrane proteins, paving the way for structural studies of this protein that is homologous to a potential drug target in HBV infection.

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing virus-infected or transfected cells via EM are the low efficiencies of infection or transfection, hindering the examination of these cells. In order to overcome this difficulty, light microscopy (LM) can be performed first to allocate the subpopulation of infected or transfected cells. Thus, taking advantage of the use of fluorescent proteins (FPs) fused to viral proteins, LM is used here to record the positions of the "positive-transfected" cells, expressing a FP and growing on a support with an alphanumeric pattern. Subsequently, cells are further processed for EM via high pressure freezing (HPF), freeze substitution (FS) and resin embedding. The ultra-rapid freezing step ensures excellent membrane preservation of the selected cells that can then be analyzed at the ultrastructural level by transmission electron microscopy (TEM). Here, a step-by-step correlative light electron microscopy (CLEM) workflow is provided, describing sample preparation, imaging and correlation in detail. The experimental design can be also applied to address many cell biology questions.

AbstractVirale Membranproteine sind vorrangige Ziele bei der Bekämpfung von Infektionen. Ihre Strukturbestimmung bleibt jedoch eine Herausforderung, sowohl in Bezug auf die Probenvorbereitung als auch auf die Methodologie der Strukturanalyse von Proteinen in einer Lipidmembranumgebung. Zellfreie Proteinherstellung und Festkörper‐NMR‐Spektroskopie sind vielversprechende Ansätze in diesem Zusammenhang, der erstere in Bezug auf das hohe Potenzial, komplexe Proteine in nativer Form herzustellen und der letztere für die Fähigkeit, Proteine in Lipiden analysieren zu können. Wir zeigen hier, dass mg‐Mengen des kleinen Hüllproteins des Enten‐Hepatitis‐B‐Virus (DHBV) mittels zellfreier Herstellung produziert werden können, und dass die Proteine zu subviralen Partikeln zusammenlagern. Protonendetektierte 2D‐NMR‐Spektren, die bei 110 kHz schneller Probenrotation um den magischen Winkel und mit <500 μg Protein aufgenommen wurden, zeigen eine Reihe von isolierten Signalen mit Linienbreiten, die vergleichbar zu denen von Modellmembranproteinen sind. Dies ebnet den Weg für strukturelle Studien dieser komplexen supramolekularen Proteinsysteme, die Homologe eines wichtigen Zielproteins für Arzneimittel in der HBV‐Infektion sind.

Reovirus replication occurs in the cytoplasm of the host cell, in virally induced mini-organelles called virus factories. On the basis of the serotype of the virus, the virus factories can manifest as filamentous (type 1 Lang strain) or globular structures (type 3 Dearing strain). The filamentous factories morphology is dependent on the microtubule cytoskeleton; however, the exact function of the microtubule network in virus replication remains unknown. Using a combination of fluorescent microscopy, electron microscopy, and tomography of high-pressure frozen and freeze-substituted cells, we determined the ultrastructural organisation of reovirus factories. Cells infected with the reovirus microtubule-dependent strain display paracrystalline arrays of progeny virions resulting from their tiered organisation around microtubule filaments. On the contrary, in cells infected with the microtubule-independent strain, progeny virions lacked organisation. Conversely to the microtubule-dependent strain, around half of the viral particles present in these viral factories did not contain genomes (genome-less particles). Complementarily, interference with the microtubule filaments in cells infected with the microtubule-dependent strain resulted in a significant increase of genome-less particle number. This decrease of genome packaging efficiency could be rescued by rerouting viral factories on the actin cytoskeleton. These findings demonstrate that the scaffolding properties of the microtubule, and not biochemical nature of tubulin, are critical determinants for reovirus efficient genome packaging. This work establishes, for the first time, a functional correlation between ultrastructural organisation of reovirus factories with genome packaging efficiency and provides novel information on how viruses coordinate assembly of progeny particles.