A unifying structural and functional model of the coronavirus replication organelle: Tracking down RNA synthesis.

Zoonotic coronavirus (CoV) infections, such as those responsible for the current severe acute respiratory syndrome-CoV 2 (SARS-CoV-2) pandemic, cause grave international public health concern. In infected cells, the CoV RNA-synthesizing machinery associates with modified endoplasmic reticulum membranes that are transformed into the viral replication organelle (RO). Although double-membrane vesicles (DMVs) appear to be a pan-CoV RO element, studies to date describe an assortment of additional CoV-induced membrane structures. Despite much speculation, it remains unclear which RO element(s) accommodate viral RNA synthesis. Here we provide detailed 2D and 3D analyses of CoV ROs and show that diverse CoVs essentially induce the same membrane modifications, including the small open double-membrane spherules (DMSs) previously thought to be restricted to gamma- and delta-CoV infections and proposed as sites of replication. Metabolic labeling of newly synthesized viral RNA followed by quantitative electron microscopy (EM) autoradiography revealed abundant viral RNA synthesis associated with DMVs in cells infected with the beta-CoVs Middle East respiratory syndrome-CoV (MERS-CoV) and SARS-CoV and the gamma-CoV infectious bronchitis virus. RNA synthesis could not be linked to DMSs or any other cellular or virus-induced structure. Our results provide a unifying model of the CoV RO and clearly establish DMVs as the central hub for viral RNA synthesis and a potential drug target in CoV infection.

Adaptive Mutations in Replicase Transmembrane Subunits Can Counteract Inhibition of Equine Arteritis Virus RNA Synthesis by Cyclophilin Inhibitors.

Previously, the cyclophilin inhibitors cyclosporine (CsA) and alisporivir (ALV) were shown to inhibit the replication of diverse RNA viruses, including arteriviruses and coronaviruses, which both belong to the order Nidovirales In this study, we aimed to identify arterivirus proteins involved in the mode of action of cyclophilin inhibitors and to investigate how these compounds inhibit arterivirus RNA synthesis in the infected cell. Repeated passaging of the arterivirus prototype equine arteritis virus (EAV) in the presence of CsA revealed that reduced drug sensitivity is associated with the emergence of adaptive mutations in nonstructural protein 5 (nsp5), one of the transmembrane subunits of the arterivirus replicase polyprotein. Introduction of singular nsp5 mutations (nsp5 Q21R, Y113H, or A134V) led to an ∼2-fold decrease in sensitivity to CsA treatment, whereas combinations of mutations further increased EAV's CsA resistance. The detailed experimental characterization of engineered EAV mutants harboring CsA resistance mutations implicated nsp5 in arterivirus RNA synthesis. Particularly, in an in vitro assay, EAV RNA synthesis was far less sensitive to CsA treatment when nsp5 contained the adaptive mutations mentioned above. Interestingly, for increased sensitivity to the closely related drug ALV, CsA-resistant nsp5 mutants required the incorporation of an additional adaptive mutation, which resided in nsp2 (H114R), another transmembrane subunit of the arterivirus replicase. Our study provides the first evidence for the involvement of nsp2 and nsp5 in the mechanism underlying the inhibition of arterivirus replication by cyclophilin inhibitors.IMPORTANCE Currently, no approved treatments are available to combat infections with nidoviruses, a group of positive-stranded RNA viruses, including important zoonotic and veterinary pathogens. Previously, the cyclophilin inhibitors cyclosporine (CsA) and alisporivir (ALV) were shown to inhibit the replication of diverse nidoviruses (both arteriviruses and coronaviruses), and they may thus represent a class of pan-nidovirus inhibitors. In this study, using the arterivirus prototype equine arteritis virus, we have established that resistance to CsA and ALV treatment is associated with adaptive mutations in two transmembrane subunits of the viral replication machinery, nonstructural proteins 2 and 5. This is the first evidence for the involvement of specific replicase subunits of arteriviruses in the mechanism underlying the inhibition of their replication by cyclophilin inhibitors. Understanding this mechanism of action is of major importance to guide future drug design, both for nidoviruses and for other RNA viruses inhibited by these compounds.

A molecular pore spans the double membrane of the coronavirus replication organelle.

Coronavirus genome replication is associated with virus-induced cytosolic double-membrane vesicles, which may provide a tailored microenvironment for viral RNA synthesis in the infected cell. However, it is unclear how newly synthesized genomes and messenger RNAs can travel from these sealed replication compartments to the cytosol to ensure their translation and the assembly of progeny virions. In this study, we used cellular cryo-electron microscopy to visualize a molecular pore complex that spans both membranes of the double-membrane vesicle and would allow export of RNA to the cytosol. A hexameric assembly of a large viral transmembrane protein was found to form the core of the crown-shaped complex. This coronavirus-specific structure likely plays a key role in coronavirus replication and thus constitutes a potential drug target.

Human VAPome Analysis Reveals MOSPD1 and MOSPD3 as Membrane Contact Site Proteins Interacting with FFAT-Related FFNT Motifs.

Membrane contact sites (MCS) are intracellular regions where two organelles come closer to exchange information and material. The majority of the endoplasmic reticulum (ER) MCS are attributed to the ER-localized tether proteins VAPA, VAPB, and MOSPD2. These recruit other proteins to the ER by interacting with their FFAT motifs. Here, we describe MOSPD1 and MOSPD3 as ER-localized tethers interacting with FFAT motif-containing proteins. Using BioID, we identify proteins interacting with VAP and MOSPD proteins and find that MOSPD1 and MOSPD3 prefer unconventional FFAT-related FFNT (two phenylalanines [FF] in a neutral tract) motifs. Moreover, VAPA/VAPB/MOSPD2 and MOSPD1/MOSPD3 assemble into two separate ER-resident complexes to interact with FFAT and FFNT motifs, respectively. Because of their ability to interact with FFNT motifs, MOSPD1 and MOSPD3 could form MCS between the ER and other organelles. Collectively, these findings expand the VAP family of proteins and highlight two separate complexes in control of interactions between intracellular compartments.

Origins of Enterovirus Replication Organelles Established by Whole-Cell Electron Microscopy.

Enterovirus genome replication occurs at virus-induced structures derived from cellular membranes and lipids. However, the origin of these replication organelles (ROs) remains uncertain. Ultrastructural evidence of the membrane donor is lacking, suggesting that the sites of its transition into ROs are rare or fleeting. To overcome this challenge, we combined live-cell imaging and serial block-face scanning electron microscopy of whole cells to capture emerging enterovirus ROs. The first foci of fluorescently labeled viral protein correlated with ROs connected to the endoplasmic reticulum (ER) and preceded the appearance of ROs stemming from the trans-Golgi network. Whole-cell data sets further revealed striking contact regions between ROs and lipid droplets that may represent a route for lipid shuttling to facilitate RO proliferation and genome replication. Our data provide direct evidence that enteroviruses use ER and then Golgi membranes to initiate RO formation, demonstrating the remarkable flexibility with which enteroviruses usurp cellular organelles.IMPORTANCE Enteroviruses are causative agents of a range of human diseases. The replication of these viruses within cells relies on specialized membranous structures termed replication organelles (ROs) that form during infection but whose origin remains elusive. To capture the emergence of enterovirus ROs, we use correlative light and serial block-face scanning electron microscopy, a powerful method to pinpoint rare events in their whole-cell ultrastructural context. RO biogenesis was found to occur first at ER and then at Golgi membranes. Extensive contacts were found between early ROs and lipid droplets (LDs), which likely serve to provide LD-derived lipids required for replication. Together, these data establish the dual origin of enterovirus ROs and the chronology of their biogenesis at different supporting cellular membranes.

Drosophila Dystrophin is required for integrity of the musculature.

Duchenne muscular dystrophy is caused by mutations in the dystrophin gene and is characterized by progressive muscle wasting. The highly conserved dystrophin gene encodes a number of protein isoforms. The Dystrophin protein is part of a large protein assembly, the Dystrophin glycoprotein complex, which stabilizes the muscle membrane during contraction and acts as a scaffold for signaling molecules. How the absence of Dystrophin results in the onset of muscular dystrophy remains unclear. Here, we have used transgenic RNA interference to examine the roles of the Drosophila Dystrophin isoforms in muscle. We previously reported that one of the Drosophila Dystrophin orthologs, the DLP2 isoform, is not required to maintain muscle integrity, but plays a role in neuromuscular homeostasis by regulating neurotransmitter release. In this report, we show that reduction of all Dystrophin isoform expression levels in the musculature does not apparently affect myogenesis or muscle attachment, but results in progressive muscle degeneration in larvae and adult flies. We find that a recently identified Dystrophin isoform, Dp117, is expressed in the musculature and is required for muscle integrity. Muscle fibers with reduced levels of Dp117 display disorganized actin-myosin filaments and the cellular hallmarks of necrosis. Our results indicate the existence of at least two possibly separate roles of dystrophin in muscle, maintaining synaptic homeostasis and preserving the structural stability of the muscle.

Disruption of Drosophila Rad50 causes pupal lethality, the accumulation of DNA double-strand breaks and the induction of apoptosis in third instar larvae.

The Rad50/Mre11/Nbs1 protein complex has a crucial role in DNA metabolism, in particular in double-strand break (DSB) repair through homologous recombination (HR). To elucidate the role of the Rad50 protein complex in DSB repair in a multicellular eukaryote, we generated a Rad50 deficient Drosophila strain by P-element mediated mutagenesis. Disruption of Rad50 causes retarded development and pupal lethality. To investigate the mechanism of pupal death, brains and wing imaginal discs from third instar larvae were studied in more detail. Wing imaginal discs from Rad50 mutant larvae displayed a 3.5-fold increase in the induction of spontaneous apoptotic cells in comparison to their heterozygous siblings. This finding correlates with increased levels of phosphorylated histone H2Av, indicating an accumulation of DSBs in Rad50 mutant larvae. A 45-fold increase in the frequency of anaphase bridges was detected in the brains of Rad50 deficient larvae, consistent with a role for Rad50 in telomere maintenance and/or replication of DNA. The induction of DSBs and defects in chromosome segregation are in agreement with a role of Drosophila Rad50 in repairing the DSBs that arise during replication.

The Drosophila melanogaster DNA Ligase IV gene plays a crucial role in the repair of radiation-induced DNA double-strand breaks and acts synergistically with Rad54.

DNA Ligase IV has a crucial role in double-strand break (DSB) repair through nonhomologous end joining (NHEJ). Most notably, its inactivation leads to embryonic lethality in mammals. To elucidate the role of DNA Ligase IV (Lig4) in DSB repair in a multicellular lower eukaryote, we generated viable Lig4-deficient Drosophila strains by P-element-mediated mutagenesis. Embryos and larvae of mutant lines are hypersensitive to ionizing radiation but hardly so to methyl methanesulfonate (MMS) or the crosslinking agent cis-diamminedichloroplatinum (cisDDP). To determine the relative contribution of NHEJ and homologous recombination (HR) in Drosophila, Lig4; Rad54 double-mutant flies were generated. Survival studies demonstrated that both HR and NHEJ have a major role in DSB repair. The synergistic increase in sensitivity seen in the double mutant, in comparison with both single mutants, indicates that both pathways partially overlap. However, during the very first hours after fertilization NHEJ has a minor role in DSB repair after exposure to ionizing radiation. Throughout the first stages of embryogenesis of the fly, HR is the predominant pathway in DSB repair. At late stages of development NHEJ also becomes less important. The residual survival of double mutants after irradiation strongly suggests the existence of a third pathway for the repair of DSBs in Drosophila.

Homodimerization of the Wnt receptor DERAILED recruits the Src family kinase SRC64B.

Ryk pseudokinase receptors act as important transducers of Wnt signals, particularly in the nervous system. Little is known, however, of their interactions at the cell surface. Here, we show that a Drosophila Ryk family member, DERAILED (DRL), forms cell surface homodimers and can also heterodimerize with the two other fly Ryks, DERAILED-2 and DOUGHNUT ON 2. DERAILED homodimerization levels increase significantly in the presence of its ligand, WNT5. In addition, DERAILED displays ligand-independent dimerization mediated by a motif in its transmembrane domain. Increased dimerization of DRL upon WNT5 binding or upon the replacement of DERAILED's extracellular domain with the immunoglobulin Fc domain results in an increased recruitment of the Src family kinase SRC64B, a previously identified downstream pathway effector. Formation of the SRC64B/DERAILED complex requires SRC64B's SH2 domain and DERAILED's PDZ-binding motif. Mutations in DERAILED's inactive tyrosine kinase-homologous domain also disrupt the formation of DERAILED/SRC64B complexes, indicating that its conformation is likely important in facilitating its interaction with SRC64B. Finally, we show that DERAILED's function during embryonic axon guidance requires its Wnt-binding domain, a putative juxtamembrane extracellular tetrabasic cleavage site, and the PDZ-binding domain, indicating that DERAILED's activation involves a complex set of events including both dimerization and proteolytic processing.

WNT5 interacts with the Ryk receptors doughnut and derailed to mediate muscle attachment site selection in Drosophila melanogaster.

In recent years a number of the genes that regulate muscle formation and maintenance in higher organisms have been identified. Studies employing invertebrate and vertebrate model organisms have revealed that many of the genes required for early mesoderm specification are highly conserved throughout evolution. Less is known about the molecules that mediate the steps subsequent to myogenesis, e. g. myotube guidance and attachment to tendon cells. We use the stereotypic pattern of the Drosophila embryonic body wall musculature in genetic approaches to identify novel factors required for muscle attachment site selection. Here, we show that Wnt5 is needed in this process. The lateral transverse muscles frequently overshoot their target attachment sites and stably attach at novel epidermal sites in Wnt5 mutant embryos. Restoration of WNT5 expression in either the muscle or the tendon cell rescues the mutant phenotype. Surprisingly, the novel attachment sites in Wnt5 mutants frequently do not express the Stripe (SR) protein which has been shown to be required for terminal tendon cell differentiation. A muscle bypass phenotype was previously reported for embryos lacking the WNT5 receptor Derailed (DRL). drl and Wnt5 mutant embryos also exhibit axon path finding errors. DRL belongs to the conserved Ryk receptor tyrosine kinase family which includes two other Drosophila orthologs, the Doughnut on 2 (DNT) and Derailed-2 (DRL-2) proteins. We generated a mutant allele of dnt and find that dnt, but not Drl-2, mutant embryos also show a muscle bypass phenotype. Genetic interaction experiments indicate that drl and dnt act together, likely as WNT5 receptors, to control muscle attachment site selection. These results extend previous findings that at least some of the molecular pathways that guide axons towards their targets are also employed for guidance of muscle fibers to their appropriate attachment sites.

Src family kinases are required for WNT5 signaling through the Derailed/RYK receptor in the Drosophila embryonic central nervous system.

Members of the RYK/Derailed family have recently been shown to regulate axon guidance in both Drosophila and mammals by acting as Wnt receptors. Little is known about how the kinase activity-deficient RYKs transduce Wnt signals. Here, we show that the non-receptor Src family tyrosine kinases, SRC64B and SRC42A, are involved in WNT5-mediated signaling through Derailed in the Drosophila embryonic central nervous system. Analysis of animals lacking SRC64B and SRC42A reveals defects in commissure formation similar to those observed in Wnt5 and derailed mutants. Reductions in SRC64B expression levels suppress a Wnt5/derailed-dependent dominant gain-of-function phenotype, and increased levels of either SRC64B or SRC42A enhance Wnt5/derailed-mediated axon commissure switching. Derailed and SRC64B form a complex, which contains catalytically active SRC64B, the formation or stability of which requires SRC64B kinase activity. Furthermore, Derailed is phosphorylated in a SRC64B-dependent manner and coexpression of Derailed and SRC64B results in the activation of SRC64B. The mammalian orthologs of Derailed and SRC64B also form complexes, suggesting that Src roles in RYK signaling are conserved. Finally, we show that coexpression of WNT5 and Derailed has no apparent effect upon TCF/LEF-dependent transcription, suggesting that the WNT5/Derailed signaling pathway is unlikely to directly regulate canonical Wnt pathway targets. Together, these findings indicate that the Src family kinases play novel roles in WNT5/Derailed-mediated signaling.