Replication Organelles Research Articles

Membrane-Associated Flavivirus Replication Complex-Its Organization and Regulation.

Flavivirus consists of a large number of arthropod-borne viruses, many of which cause life-threatening diseases in humans. A characteristic feature of flavivirus infection is to induce the rearrangement of intracellular membrane structure in the cytoplasm. This unique membranous structure called replication organelle is considered as a microenvironment that provides factors required for the activity of the flaviviral replication complex. The replication organelle serves as a place to coordinate viral RNA amplification, protein translation, and virion assembly and also to protect the viral replication complex from the cellular immune defense system. In this review, we summarize the current understanding of how the formation and function of membrane-associated flaviviral replication organelle are regulated by cellular factors.

Enterovirus 71 Induces INF2 Cleavage via Activated Caspase-2 in Infected RD Cells.

Enterovirus 71 (EV71) is the major causative pathogen of hand, foot, and mouth disease. The lack of understanding of the virus’s pathogenesis hinders the development of anti-virus drugs and the control of EV71 infection. Our previous studies have demonstrated that both mitochondria and endoplasmic reticulum (ER) were altered significantly in EV71 infected cells, but the mechanism is still unclear. In this study, we investigated the effects of EV71 infection on the expression of INF2, a key regulator factor in ER-Mitochondria communication and mitochondrial fission. We found that INF2 was cleaved in EV71 infected RD cells. The INF2 cleavage occurred at Aspartic 1,051 of INF2 and is mediated by activated caspases, predominantly by activated caspase-2. The subcellular localization of INF2 and caspase-2 was significantly altered in infected cells. We speculate that caspase-2-mediated INF2 cleavage is involved in forming viral replication organelles (ROs) and is a positive feedback regulatory mechanism of mitochondrial disorders caused by EV71 infection.

Electrostatic Interaction Between NS1 and Negatively Charged Lipids Contributes to Flavivirus Replication Organelles Formation.

Flavivirus replication occurs in membranous replication compartments, also known as replication organelles (ROs) derived from the host ER membrane. Our previous study showed that the non-structural (NS) protein 1 (NS1) is the essential factor for RO creation by hydrophobic insertion into the ER membrane. Here, we found that the association of NS1 with the membrane can be facilitated by the electrostatic interaction between NS1 and negatively charged lipids. NS1 binds to a series of negatively charged lipids, including PI4P, and a positively charged residue, R31, located on the membrane-binding face of NS1, plays important roles in this interaction. The NS1 R31E mutation significantly impairs NS1 association with negatively charged membrane and its ER remodeling ability in the cells. To interfere with the electrostatic interaction between NS1 and negatively charged lipids, intracellular phosphatidylinositol phosphates (PIPs) level was downregulated by the overexpression of Sac1 or treatment with PI3K and PI4K inhibitors to attenuate flavivirus replication. Our findings emphasize the importance of electrostatic interaction between NS1 and negatively charged lipids in flavivirus RO formation.

Light and electron microscopy imaging unveils new aspects of the antiviral capacity of silver nanoparticles in bunyavirus-infected cells

Drug repurposing is an important source of new antivirals because many compounds used to treat a variety of pathologies also hamper viral infections. Habitually, silver nanoparticles (AgNPs) have been used to treat bacterial and fungal infections and their antiviral properties have been also reported. In this work, we have studied the antiviral capacity of AgNPs in cells infected with Bunyamwera virus (BUNV), the prototype of the Bunyavirales order. This group of viruses contains important pathogens for humans, animals and plants. Incubation of BUNV-infected Vero cells with non-toxic concentrations of AgNPs, reduced the production of extracellular infectious viruses in up to three orders of magnitude. With a combination of imaging techniques, we have visualized the intracellular distribution of AgNPs in mock- and BUNV-infected cells and studied their effects on intracellular organelles. In mock-infected cells and at short times post-incubation, AgNPs were detected inside nuclei and mitochondria by transmission electron microscopy (TEM). At long times post-treatment, they accumulated inside lysosome-like organelles. Cell compartments did not exhibit any appreciable ultrastructural alterations after incubation with AgNPs. In BUNV-infected cells, AgNPs attached to extracellular virions, that showed a disrupted morphology. Inside cells, they were detected inside the nucleus, in mitochondria and around characteristic Golgi-associated, single-membrane spherules. These membranous structures are the replication organelles (ROs) of bunyaviruses and contain active viral replication complexes (VRCs). Compared to normal spherules that are round, compact and have an electron-dense core, spherules in AgNPs-treated cells were deformed and their core was electron-lucent. Interestingly, in BUNV-infected cells treated with the typical antiviral ribavirin (RBV), spherules with VRCs exhibit also an anomalous morphology and an electron-lucent core. Both AgNPs and RBV might interfere with BUNV-induced dismantling of cell nucleoli and with the intercellular propagation of large groups of virions, a mechanism of BUNV transmission observed for the first time in cultured cells. Our results point to silver nanoparticles as good candidates for antiviral therapy, either alone or in combination with other antiviral drugs, such as RBV-related compounds.

Viral pores are everywhere

Jones etal. (2021) and Zhang etal. (2021) reveal by cryo-EM the oligomeric crown-like structure formed by a membrane-associated Chikungunya virus replication protein that gates the export of newly synthesized viral RNA from viral replication organelles.

Hepatitis C Viral Replication Complex.

The life cycle of the hepatitis C virus (HCV) can be divided into several stages, including viral entry, protein translation, RNA replication, viral assembly, and release. HCV genomic RNA replication occurs in the replication organelles (RO) and is tightly linked to ER membrane alterations containing replication complexes (proteins NS3 to NS5B). The amplification of HCV genomic RNA could be regulated by the RO biogenesis, the viral RNA structure (i.e., cis-acting replication elements), and both viral and cellular proteins. Studies on HCV replication have led to the development of direct-acting antivirals (DAAs) targeting the replication complex. This review article summarizes the viral and cellular factors involved in regulating HCV genomic RNA replication and the DAAs that inhibit HCV replication.

Co-opting of nonATP-generating glycolytic enzymes for TBSV replication

Positive-strand RNA viruses build viral replication organelles (VROs) with the help of co-opted host factors. The energy requirement of intensive viral replication processes is less understood. Previous studies on tomato bushy stunt virus (TBSV) showed that tombusviruses hijack two ATP-producing glycolytic enzymes to produce ATP locally within VROs. In this work, we performed a cDNA library screen with Arabidopsis thaliana proteins and the TBSV p33 replication protein. The p33 - plant interactome contained highly conserved glycolytic proteins. We find that the glycolytic Hxk2 hexokinase, Eno2 phosphopyruvate hydratase and Fba1 fructose 1,6-bisphosphate aldolase are critical for TBSV replication in yeast or in a cell-free replicase reconstitution assay. The recruitment of Fba1 is important for the local production of ATP within VROs. Altogether, our data support the model that TBSV recruits and compartmentalizes possibly most members of the glycolytic pathway. This might allow TBSV to avoid competition with the host for ATP.

Modeling the complete kinetics of coxsackievirus B3 reveals human determinants of host-cell feedback.

Complete kinetic models are pervasive in chemistry but lacking in biological systems. We encoded the complete kinetics of infection for coxsackievirus B3 (CVB3), a compact and fast-acting RNA virus. The model consists of separable, detailed modules describing viral binding-delivery, translation-replication, and encapsidation. Specific module activities are dampened by the type I interferon response to viral double-stranded RNAs (dsRNAs), which is itself disrupted by viral proteinases. The experimentally validated kinetics uncovered that cleavability of the dsRNA transducer mitochondrial antiviral signaling protein (MAVS) becomes a stronger determinant of viral outcomes when cells receive supplemental interferon after infection. Cleavability is naturally altered in humans by a common MAVS polymorphism, which removes a proteinase-targeted site but paradoxically elevates CVB3 infectivity. These observations are reconciled with a simple nonlinear model of MAVS regulation. Modeling complete kinetics is an attainable goal for small, rapidly infecting viruses and perhaps viral pathogens more broadly. A record of this paper's transparent peer review process is included in the Supplemental information.

Exploiting Connections for Viral Replication.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the COVID-19 (coronavirus disease 2019) pandemic, is a positive strand RNA (+RNA) virus. Like other +RNA viruses, SARS-CoV-2 is dependent on host cell metabolic machinery to survive and replicate, remodeling cellular membranes to generate sites of viral replication. Viral RNA-containing double-membrane vesicles (DMVs) are a striking feature of +RNA viral replication and are abundant in SARS-CoV-2–infected cells. Their generation involves rewiring of host lipid metabolism, including lipid biosynthetic pathways. Viruses can also redirect lipids from host cell organelles; lipid exchange at membrane contact sites, where the membranes of adjacent organelles are in close apposition, has been implicated in the replication of several +RNA viruses. Here we review current understanding of DMV biogenesis. With a focus on the exploitation of contact site machinery by +RNA viruses to generate replication organelles, we discuss evidence that similar mechanisms support SARS-CoV-2 replication, protecting its RNA from the host cell immune response.

An Overview of Immune Evasion Strategies of DNA and RNA Viruses.

A successful viral infection is due to the effective evasion of viruses of the immune system. The entry of viruses is usually detected by different cellular receptors including PRRs. Recognition of the viral genome leads to the production of interferons through a signaling stream. This review article provides brief information regarding escape mechanisms of DNA and RNA viruses from the host immune system. These strategies include viral endonuclease activity, cap snatching of host mRNA, the formation of replication organelles, stress granule formation, membrane modifications, action of proteases, and evasion from ISGs. Moreover, the strategies of DNA viruses to inhibit immune responses include subversion of mRNA, transcriptional factors, adaptor proteins, PRRs, evasion from T lymphocytes, genomic diversity, theft or seizure of host defense proteins, imitation of host factors like affecting cytokines and chemokines of the host, suppression or inhibition of apoptosis, and proteasomal degradation of host antiviral proteins by DNA viruses. The knowledge of these mechanisms is pivotal to understanding different methodologies that viruses have created to escape antiviral cellular reactions of the host as well as virus-host interactions and the origin of viral pathogenesis. Also, this knowledge is significant for the design of gene targeting vectors, antiviral vaccines, and the development of effective treatments directed against DNA and RNA viruses.

Picornaviruses: A View from 3A.

Picornaviruses are comprised of a positive-sense RNA genome surrounded by a protein shell (or capsid). They are ubiquitous in vertebrates and cause a wide range of important human and animal diseases. The genome encodes a single large polyprotein that is processed to structural (capsid) and non-structural proteins. The non-structural proteins have key functions within the viral replication complex. Some, such as 3Dpol (the RNA dependent RNA polymerase) have conserved functions and participate directly in replicating the viral genome, whereas others, such as 3A, have accessory roles. The 3A proteins are highly divergent across the Picornaviridae and have specific roles both within and outside of the replication complex, which differ between the different genera. These roles include subverting host proteins to generate replication organelles and inhibition of cellular functions (such as protein secretion) to influence virus replication efficiency and the host response to infection. In addition, 3A proteins are associated with the determination of host range. However, recent observations have challenged some of the roles assigned to 3A and suggest that other viral proteins may carry them out. In this review, we revisit the roles of 3A in the picornavirus life cycle. The 3AB precursor and mature 3A have distinct functions during viral replication and, therefore, we have also included discussion of some of the roles assigned to 3AB.

Japanese encephalitis virus manipulates lysosomes membrane for RNA replication and utilizes autophagy components for intracellular growth

Japanese encephalitis virus is absolutely dependent on their host cells and has evolved various strategies to manipulate the cellular secretory pathways for viral replication. However, how cellular secretory pathways are hijacked, and the origin of the viral vesicles remains elusive during JEV replication. Here we show how JEV manipulates multiple components of the cellular secretory pathway, including autophagic machinery, to generate a superior environment for genome replication. We utilized double-strand RNA antibodies to label JEV RNA complex seeking the viral replication compartments and found that JEV genome replication takes place in lysosomes (LAMP1), not in autophagosomes (LC3). Subsequently, in situ hybridization results showed that viral RNAs (vRNAs) of JEV strongly colocalized with LAMP1. What surprised us was that JEV vRNAs markedly colocalized with LC3, indicating that autophagy plays an active role in JEV replication. Interestingly, we found that JEV utilized autophagic components for intracellular growth in an autophagy-dependent manner and the fusion of autophagosome-lysosome plays a positive role in JEV post-RNA replication processes. Collectively, our findings demonstrate that JEV can manipulate cellular secretory pathway to form genome replication organelles and exploit autophagy components for intracellular growth, providing new insights into the life cycle of JEV and uncovering an attractive target for antiviral drugs.

Multiscale Electron Microscopy for the Study of Viral Replication Organelles.

During infection with positive-strand RNA viruses, viral RNA synthesis associates with modified intracellular membranes that form unique and captivating structures in the cytoplasm of the infected cell. These viral replication organelles (ROs) play a key role in the replicative cycle of important human pathogens like coronaviruses, enteroviruses, or flaviviruses. From their discovery to date, progress in our understanding of viral ROs has closely followed new developments in electron microscopy (EM). This review gives a chronological account of this progress and an introduction to the different EM techniques that enabled it. With an ample repertoire of imaging modalities, EM is nowadays a versatile technique that provides structural and functional information at a wide range of scales. Together with well-established approaches like electron tomography or labeling methods, we examine more recent developments, such as volume scanning electron microscopy (SEM) and in situ cryotomography, which are only beginning to be applied to the study of viral ROs. We also highlight the first cryotomography analyses of viral ROs, which have led to the discovery of macromolecular complexes that may serve as RO channels that control the export of newly-made viral RNA. These studies are key first steps towards elucidating the macromolecular complexity of viral ROs.

ADAM15 Participates in Tick-Borne Encephalitis Virus Replication.

Tick-borne encephalitis virus (TBEV), a major tick-borne viral pathogen of humans, is known to cause neurological diseases such as meningitis, encephalitis, and meningoencephalitis. However, the life cycle and pathogenesis of TBEV are not well understood. Here, we show that the knockdown or knockout of ADAM15 (a disintegrin and metalloproteinase 15), a host protein involved in neuroblastoma diseases, leads to TBEV replication and assembly defects. We characterized the disintegrin domain in ADAM15 and found that the ADAM15 subcellular localization was changed following TBEV infection. RNA interference (RNAi) screen analysis confirmed ADAM's nonredundant functions and identified a specific role for ADAM15 in TBEV infection. An RNA-sequencing analysis was also conducted to understand the causal link between TBEV infection and the cellular endomembrane network, namely, the generation of replication organelles promoting viral genome replication and virus production. Our data demonstrated that TBEV infection changes ADAM15 cellular localization, which contributes to membrane reorganization and viral replication.IMPORTANCE Tick populations are increasing, and their geographic ranges are expanding. Increases in tick-borne disease prevalence and transmission are important public health issues. Tick-borne encephalitis virus (TBEV) often results in meningitis, encephalitis, and meningoencephalitis. TBEV causes clinical disease in more than 20,000 humans in Europe and Asia per year. An increased incidence of TBE has been noted in Europe and Asia, as a consequence of climate and socioeconomic changes. The need to investigate the mechanism(s) of interaction between the virus and the host factors is apparent, as it will help us to understand the roles of host factors in the life cycle of TBEV. The significance of our research is in identifying the ADAM15 for TBEV replication, which will greatly enhance our understanding of TBEV life cycle and highlight a target for pharmaceutical consideration.

Return of the Neurotropic Enteroviruses: Co-Opting Cellular Pathways for Infection.

Enteroviruses are among the most common human infectious agents. While infections are often mild, the severe neuropathogenesis associated with recent outbreaks of emerging non-polio enteroviruses, such as EV-A71 and EV-D68, highlights their continuing threat to public health. In recent years, our understanding of how non-polio enteroviruses co-opt cellular pathways has greatly increased, revealing intricate host–virus relationships. In this review, we focus on newly identified mechanisms by which enteroviruses hijack the cellular machinery to promote their replication and spread, and address their potential for the development of host-directed therapeutics. Specifically, we discuss newly identified cellular receptors and their contribution to neurotropism and spread, host factors required for viral entry and replication, and recent insights into lipid acquisition and replication organelle biogenesis. The comprehensive knowledge of common cellular pathways required by enteroviruses could expose vulnerabilities amenable for host-directed therapeutics against a broad spectrum of enteroviruses. Since this will likely include newly arising strains, it will better prepare us for future epidemics. Moreover, identifying host proteins specific to neurovirulent strains may allow us to better understand factors contributing to the neurotropism of these viruses.

Rab1-AMPylation by Legionella DrrA is allosterically activated by Rab1

Legionella pneumophila infects eukaryotic cells by forming a replicative organelle – the Legionella containing vacuole. During this process, the bacterial protein DrrA/SidM is secreted and manipulates the activity and post-translational modification (PTM) states of the vesicular trafficking regulator Rab1. As a result, Rab1 is modified with an adenosine monophosphate (AMP), and this process is referred to as AMPylation. Here, we use a chemical approach to stabilise low-affinity Rab:DrrA complexes in a site-specific manner to gain insight into the molecular basis of the interaction between the Rab protein and the AMPylation domain of DrrA. The crystal structure of the Rab:DrrA complex reveals a previously unknown non-conventional Rab-binding site (NC-RBS). Biochemical characterisation demonstrates allosteric stimulation of the AMPylation activity of DrrA via Rab binding to the NC-RBS. We speculate that allosteric control of DrrA could in principle prevent random and potentially cytotoxic AMPylation in the host, thereby perhaps ensuring efficient infection by Legionella.

Valosin‐containing protein ATPase activity regulates the morphogenesis of Zika virus replication organelles and virus‐induced cell death

With no available therapies, infections with Zika virus (ZIKV) constitute a major public health concern as they can lead to congenital microcephaly. In order to generate an intracellular environment favourable to viral replication, ZIKV induces endomembrane remodelling and the morphogenesis of replication factories via enigmatic mechanisms. In this study, we identified the AAA+ type ATPase valosin-containing protein (VCP) as a cellular interaction partner of ZIKV non-structural protein 4B (NS4B). Importantly, its pharmacological inhibition as well as the expression of a VCP dominant-negative mutant impaired ZIKV replication. In infected cells, VCP is relocalised to large ultrastructures containing both NS4B and NS3, which are reminiscent of dengue virus convoluted membranes. Moreover, short treatment with the VCP inhibitors NMS-873 or CB-5083 drastically decreased the abundance and size of ZIKV-induced convoluted membranes. Furthermore, NMS-873 treatment inhibited ZIKV-induced mitochondria elongation previously reported to be physically and functionally linked to convoluted membranes in case of the closely related dengue virus. Finally, VCP inhibition resulted in enhanced apoptosis of ZIKV-infected cells strongly suggesting that convoluted membranes limit virus-induced cytopathic effects. Altogether, this study identifies VCP as a host factor required for ZIKV life cycle and more precisely, for the maintenance of viral replication factories. Our data further support a model in which convoluted membranes regulate ZIKV life cycle by impacting on mitochondrial functions and ZIKV-induced death signals in order to create a cytoplasmic environment favourable to viral replication.

Zika virus replication in glioblastoma cells: electron microscopic tomography shows 3D arrangement of endoplasmic reticulum, replication organelles, and viral ribonucleoproteins

Structural changes of two patient-derived glioblastoma cell lines after Zika virus infection were investigated using scanning transmission electron tomography on high-pressure-frozen, freeze-substituted samples. In Zika-virus-infected cells, Golgi structures were barely visible under an electron microscope, and viral factories appeared. The cytosol outside of the viral factories resembled the cytosol of uninfected cells. The viral factories contained largely deranged endoplasmic reticulum (ER), filled with many so-called replication organelles consisting of a luminal vesicle surrounded by the ER membrane. Viral capsids were observed in the vicinity of the replication organelles (cell line #12537 GB) or in ER cisternae at large distance from the replication organelles (cell line #15747 GB). Near the replication organelles, we observed many about 100-nm-long filaments that may represent viral ribonucleoprotein complexes (RNPs), which consist of the RNA genome and N protein oligomers. In addition, we compared Zika-virus-infected cells with cells infected with a phlebovirus (sandfly fever Turkey virus). Zika virions are formed in the ER, whereas phlebovirus virions are assembled in the Golgi apparatus. Our findings will help to understand the replication cycle in the virus factories and the building of the replication organelles in glioblastoma cells.

The retromer is co-opted to deliver lipid enzymes for the biogenesis of lipid-enriched tombusviral replication organelles

Biogenesis of viral replication organelles (VROs) is critical for replication of positive-strand RNA viruses. In this work, we demonstrate that tomato bushy stunt virus (TBSV) and the closely related carnation Italian ringspot virus (CIRV) hijack the retromer to facilitate building VROs in the surrogate host yeast and in plants. Depletion of retromer proteins, which are needed for biogenesis of endosomal tubular transport carriers, strongly inhibits the peroxisome-associated TBSV and the mitochondria-associated CIRV replication in yeast and in planta. In vitro reconstitution revealed the need for the retromer for the full activity of the viral replicase. The viral p33 replication protein interacts with the retromer complex, including Vps26, Vps29, and Vps35. We demonstrate that TBSV p33-driven retargeting of the retromer into VROs results in delivery of critical retromer cargoes, such as 1) Psd2 phosphatidylserine decarboxylase, 2) Vps34 phosphatidylinositol 3-kinase (PI3K), and 3) phosphatidylinositol 4-kinase (PI4Kα-like). The recruitment of these cellular enzymes by the co-opted retromer is critical for de novo production and enrichment of phosphatidylethanolamine phospholipid, phosphatidylinositol-3-phosphate [PI(3)P], and phosphatidylinositol-4-phosphate [PI(4)P] phosphoinositides within the VROs. Co-opting cellular enzymes required for lipid biosynthesis and lipid modifications suggest that tombusviruses could create an optimized lipid/membrane microenvironment for efficient VRO assembly and protection of the viral RNAs during virus replication. We propose that compartmentalization of these lipid enzymes within VROs helps tombusviruses replicate in an efficient milieu. In summary, tombusviruses target a major crossroad in the secretory and recycling pathways via coopting the retromer complex and the tubular endosomal network to build VROs in infected cells.

Key interplay between the co-opted sorting nexin-BAR proteins and PI3P phosphoinositide in the formation of the tombusvirus replicase.

Positive-strand RNA viruses replicate in host cells by forming large viral replication organelles, which harbor numerous membrane-bound viral replicase complexes (VRCs). In spite of its essential role in viral replication, the biogenesis of the VRCs is not fully understood. The authors identified critical roles of cellular membrane-shaping proteins and PI(3)P (phosphatidylinositol 3-phosphate) phosphoinositide, a minor lipid with key functions in endosomal vesicle trafficking and autophagosome biogenesis, in VRC formation for tomato bushy stunt virus (TBSV). The authors show that TBSV co-opts the endosomal SNX-BAR (sorting nexin with Bin/Amphiphysin/Rvs- BAR domain) proteins, which bind to PI(3)P and have membrane-reshaping function during retromer tubular vesicle formation, directly into the VRCs to boost progeny viral RNA synthesis. We find that the viral replication protein-guided recruitment and pro-viral function of the SNX-BAR proteins depends on enrichment of PI(3)P at the site of viral replication. Depletion of SNX-BAR proteins or PI(3)P renders the viral double-stranded (ds)RNA replication intermediate RNAi-sensitive within the VRCs in the surrogate host yeast and in planta and ribonuclease-sensitive in cell-free replicase reconstitution assays in yeast cell extracts or giant unilamellar vesicles (GUVs). Based on our results, we propose that PI(3)P and the co-opted SNX-BAR proteins are coordinately exploited by tombusviruses to promote VRC formation and to play structural roles and stabilize the VRCs during viral replication. Altogether, the interplay between the co-opted SNX-BAR membrane-shaping proteins, PI(3)P and the viral replication proteins leads to stable VRCs, which provide the essential protection of the viral RNAs against the host antiviral responses.