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.

Autophagy is a cellular response to starvation which generates autophagosomes to carry cellular organelles and long-lived proteins to lysosomes for degradation. Degradation through autophagy can provide an innate defence against virus infection, or conversely autophagosomes can promote infection by facilitating assembly of replicase proteins. We demonstrate that the avian coronavirus, Infectious Bronchitis Virus (IBV) activates autophagy. A screen of individual IBV non-structural proteins (nsps) showed that autophagy was activated by IBV nsp6. This property was shared with nsp6 of mammalian coronaviruses Mouse Hepatitis Virus, and Severe Acute Respiratory Syndrome Virus, and the equivalent nsp5-7 of the arterivirus Porcine Reproductive and Respiratory Syndrome Virus. These multiple-spanning transmembrane proteins located to the endoplasmic reticulum (ER) where they generated Atg5 and LC3II-positive vesicles, and vesicle formation was dependent on Atg5 and class III PI3 kinase. The vesicles recruited double FYVE-domain containing protein (DFCP) indicating localised concentration of phosphatidylinositol 3 phosphate, and therefore shared many features with omegasomes formed from the ER in response to starvation. Omegasomes induced by viral nsp6 matured into autophagosomes that delivered LC3 to lysosomes and therefore recruited and recycled the proteins needed for autophagosome nucleation, expansion, cellular trafficking and delivery of cargo to lysosomes. The coronavirus nsp6 proteins activated omegasome and autophagosome formation independently of starvation, but activation did not involve direct inhibition of mTOR signalling, activation of sirtuin1 or induction of ER stress.

Replication of positive-sense RNA viruses is associated with the rearrangement of cellular membranes. Previous work on the infection of tissue culture cell lines with the betacoronaviruses mouse hepatitis virus and severe acute respiratory syndrome coronavirus (SARS-CoV) showed that they generate double-membrane vesicles (DMVs) and convoluted membranes as part of a reticular membrane network. Here we describe a detailed study of the membrane rearrangements induced by the avian gammacoronavirus infectious bronchitis virus (IBV) in a mammalian cell line but also in primary avian cells and in epithelial cells of ex vivo tracheal organ cultures. In all cell types, structures novel to IBV infection were identified that we have termed zippered endoplasmic reticulum (ER) and spherules. Zippered ER lacked luminal space, suggesting zippering of ER cisternae, while spherules appeared as uniform invaginations of zippered ER. Electron tomography showed that IBV-induced spherules are tethered to the zippered ER and that there is a channel connecting the interior of the spherule with the cytoplasm, a feature thought to be necessary for sites of RNA synthesis but not seen previously for membrane rearrangements induced by coronaviruses. We also identified DMVs in IBV-infected cells that were observed as single individual DMVs or were connected to the ER via their outer membrane but not to the zippered ER. Interestingly, IBV-induced spherules strongly resemble confirmed sites of RNA synthesis for alphaviruses, nodaviruses, and bromoviruses, which may indicate similar strategies of IBV and these diverse viruses for the assembly of RNA replication complexes.IMPORTANCE All positive-sense single-stranded RNA viruses induce rearranged cellular membranes, providing a platform for viral replication complex assembly and protecting viral RNA from cellular defenses. We have studied the membrane rearrangements induced by an important poultry pathogen, the gammacoronavirus infectious bronchitis virus (IBV). Previous work studying closely related betacoronaviruses identified double-membrane vesicles (DMVs) and convoluted membranes (CMs) derived from the endoplasmic reticulum (ER) in infected cells. However, the role of DMVs and CMs in viral RNA synthesis remains unclear because these sealed vesicles lack a means of delivering viral RNA to the cytoplasm. Here, we characterized structures novel to IBV infection: zippered ER and small vesicles tethered to the zippered ER termed spherules. Significantly, spherules contain a channel connecting their interior to the cytoplasm and strongly resemble confirmed sites of RNA synthesis for other positive-sense RNA viruses, making them ideal candidates for the site of IBV RNA synthesis.

SARS Coronavirus 2 (SARS-CoV-2) emerged in late 2019, leading to the Coronavirus Disease 2019 (COVID-19) pandemic that continues to cause significant global mortality in human populations. Given its sequence similarity to SARS-CoV, as well as related coronaviruses circulating in bats, SARS-CoV-2 is thought to have originated in Chiroptera species in China. However, whether the virus spread directly to humans or through an intermediate host is currently unclear, as is the potential for this virus to infect companion animals, livestock, and wildlife that could act as viral reservoirs. Using a combination of surrogate entry assays and live virus, we demonstrate that, in addition to human angiotensin-converting enzyme 2 (ACE2), the Spike glycoprotein of SARS-CoV-2 has a broad host tropism for mammalian ACE2 receptors, despite divergence in the amino acids at the Spike receptor binding site on these proteins. Of the 22 different hosts we investigated, ACE2 proteins from dog, cat, and cattle were the most permissive to SARS-CoV-2, while bat and bird ACE2 proteins were the least efficiently used receptors. The absence of a significant tropism for any of the 3 genetically distinct bat ACE2 proteins we examined indicates that SARS-CoV-2 receptor usage likely shifted during zoonotic transmission from bats into people, possibly in an intermediate reservoir. Comparison of SARS-CoV-2 receptor usage to the related coronaviruses SARS-CoV and RaTG13 identified distinct tropisms, with the 2 human viruses being more closely aligned. Finally, using bioinformatics, structural data, and targeted mutagenesis, we identified amino acid residues within the Spike–ACE2 interface, which may have played a pivotal role in the emergence of SARS-CoV-2 in humans. The apparently broad tropism of SARS-CoV-2 at the point of viral entry confirms the potential risk of infection to a wide range of companion animals, livestock, and wildlife.

bioRxiv preprint DMVs would pose the conundrum of how metabolites and newly made genomic and 64 subgenomic mRNAs could be transported across the double-lipid bilayer. Nevertheless, 65 experimental evidence for the location of vRNA synthesis within the RO was lacking. 66 Importantly, dsRNA is not a bona fide marker for vRNA synthesis as it may no longer be 67 associated with the active enzymatic replication complexes in which most of the 16 viral non-68 structural proteins (nsps) come together. Thus, the possibility of vRNA synthesis taking place 69 in alternative locations, like the convoluted membranes (CM) that are also prominent 70 elements of the beta-CoV RO [15, 20, 21], was entirely possible and started to attract 71 attention. Notably, DMVs can be also formed in the absence of vRNA synthesis by 72 expression of key transmembrane nsps [22-26]. Moreover, several studies suggested a lack of 73 direct correlation between the number of DMVs and the level of CoV replication in the 74 infected cell [27, 28]. 75 The interpretation of the CoV RO structure and function was further compounded by the 76 discovery of different RO elements, never reported in beta-CoV infections, that set apart 77 other distantly-related CoVs genera. In particular, zippered ER (instead of CM) and double-78 membrane spherules (DMSs) were detected for the avian gamma-CoV infectious bronchitis 79 virus (IBV) [29] and, recently, for the porcine deltacoronavirus [30]. The size and topology 80 of these DMSs, invaginations in the zippered ER, were remarkably similar to those of the 81 spherular invaginations induced by other +RNA viruses and, consequently, DMSs were 82 suggested to be sites of vRNA synthesis [29, 31]. 83 In this work, we provide an in-depth analysis of the structure and function of the RO induced 84 by members of different CoV genera, with a special focus on beta-CoVs like SARS-CoV and 85 MERS-CoV (Middle East respiratory syndrome-CoV) [32, 33]. While the SARS-CoV 86 outbreak was contained in 2003, MERS-CoV continues to pose a serious zoonotic threat to 87 human health since 2012 : bioRxiv preprint (right) of a representative area containing the different types of MERS-CoV-induced membrane 130 modifications: DMSs (orange), convoluted membranes (CM, blue) and DMVs (yellow and lilac, outer 131 and inner membranes). The model also highlights ER membranes (green) and a vesicle (silver) 132 containing new virions (pink). (See also S1 Video). (C) Comparison of DMSs and virions 133 (arrowheads in left and right panels, respectively) in enlarged views of tomographic slices from the 134 regions boxed in (B). The DMSs are similar in size but distinct in appearance from newly-formed 135 MERS-CoV particles. (D) Whisker plots of the size distribution of DMSs (n=58), virions (n=28) and 136 DMVs (n=109), as measured from the tomograms. DMSs and virions have a comparable size (median 137 diameter, 80 nm), while the median diameter of the DMVs is 247 nm. (E) Models and tomographic 138

ABSTRACT:Hereditary spastic paraplegia (HSP) describes a heterogeneous group of inherited neurodegenerative disorders in which the cardinal pathological feature is upper motor neurone degeneration leading to progressive spasticity and weakness of the lower limbs. Using samples from a large Omani family we recently mapped a gene for a novel autosomal recessive form of HSP (SPG35) in which the spastic paraplegia was associated with intellectual disability and seizures. Magnetic resonance imaging of the brain of SPG35 patients showed white matter abnormalities suggestive of a leukodystrophy. Here we report homozygous mutations in the fatty acid 2-hydroxylase gene (FA2H) in the original family used to define the SPG35 locus (p.Arg235Cys) as well as in a previously unreported Pakistani family with a similar phenotype (p.Arg53_Ile58del). Measurement of enzyme activity in vitro revealed significantly reduced enzymatic function of FA2H associated with these mutations. These results demonstrate that mutations in FA2H are associated with SPG35, and that abnormal hydroxylation of myelin galactocerebroside lipid components can lead to a severe progressive phenotype, with a clinical presentation of complicated HSP and radiological features of leukodystrophy. ©2010 Wiley-Liss, Inc.

Coronaviruses from both the Alphacoronavirus and Betacoronavirus genera interfere with the type I interferon (IFN) response in various ways, ensuring the limited activation of the IFN response in most cell types. Of the gammacoronaviruses that mainly infect birds, little is known about the activation of the host immune response. We show that the prototypical Gammacoronavirus, infectious bronchitis virus (IBV), induces a delayed activation of the IFN response in primary renal cells, tracheal epithelial cells, and a chicken cell line. In fact, Ifn␤ expression is delayed with respect to the peak of viral replication and the accompanying accumulation of double-stranded RNA (dsRNA). In addition, we demonstrate that MDA5 is the primary sensor for Gammacoronavirus infections in chicken cells. Furthermore, we provide evidence that accessory proteins 3a and 3b of IBV modulate the response at the transcriptional and translational levels. Finally, we show that, despite the lack of activation of the IFN response during the early phase of IBV infection, the signaling of nonself dsRNA through both MDA5 and TLR3 remains intact in IBVinfected cells. Taken together, this study provides the first comprehensive analysis of host-virus interactions of a Gammacoronavirus with avian innate immune responses. IMPORTANCEOur results demonstrate that IBV has evolved multiple strategies to avoid the activation of the type I interferon response. Taken together, the present study closes a gap in the understanding of host-IBV interaction and paves the way for further characterization of the mechanisms underlying immune evasion strategies as well as the pathogenesis of gammacoronaviruses. Coronaviruses constitute a large family of positive-stranded RNA viruses and cause a range of human and veterinary diseases. Infectious bronchitis virus (IBV) is the prototype avian coronavirus from the Gammacoronavirus genus and the causative agent of a highly contagious respiratory disease of major economic importance to the poultry industry (1). IBV enters the avian host through the respiratory tract, where it causes the destruction of the epithelium, leading to respiratory distress and initiation of secondary bacterial infections. Depending on the strain, IBV also can spread to other epithelial surfaces, such as the gastrointestinal tract, the kidneys, and the oviduct, with the latter causing problems in egg production and quality (1-6). Contrary to coronaviruses from the Alphacoronavirus and Betacoronavirus genera, including human coronavirus HCoV-229E, severe acute respiratory syndrome (SARS-CoV), Middle East respiratory syndrome (MERS-CoV), and mouse hepatitis virus (MHV), very little is known about how gammacoronaviruses, including IBV, evade or interfere with the innate immune responses of their host.Innate immune responses consist of a network of antimicrobial mechanisms, of which the type I interferon (IFN) response is an essential defense mechanism against viruses. Typically, the type I IFN response, here referred to as the IFN response, is...

Coronaviruses are single stranded, positive sense RNA viruses, which induce the rearrangement of cellular membranes upon infection of a host cell. This provides the virus with a platform for the assembly of viral replication complexes, improving efficiency of RNA synthesis. The membranes observed in coronavirus infected cells include double membrane vesicles. By nature of their double membrane, these vesicles resemble cellular autophagosomes, generated during the cellular autophagy pathway. In addition, coronavirus infection has been demonstrated to induce autophagy. Here we review current knowledge of coronavirus induced membrane rearrangements and the involvement of autophagy or autophagy protein microtubule associated protein 1B light chain 3 (LC3) in coronavirus replication.