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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread globally, and the number of worldwide cases continues to rise. The zoonotic origins of SARS-CoV-2 and its intermediate and potential spillback host reservoirs, besides humans, remain largely unknown. Because of ethical and experimental constraints and more important, to reduce and refine animal experimentation, we used our repository of well-differentiated airway epithelial cell (AEC) cultures from various domesticated and wildlife animal species to assess their susceptibility to SARS-CoV-2. We observed that SARS-CoV-2 replicated efficiently only in monkey and cat AEC culture models. Whole-genome sequencing of progeny viruses revealed no obvious signs of nucleotide transitions required for SARS-CoV-2 to productively infect monkey and cat AEC cultures. Our findings, together with previous reports of human-to-animal spillover events, warrant close surveillance to determine the potential role of cats, monkeys, and closely related species as spillback reservoirs for SARS-CoV-2.

Infection control instructions call for use of alcohol-based hand rub solutions to inactivate severe acute respiratory syndrome coronavirus 2. We determined the virucidal activity of World Health Organization-recommended hand rub formulations, at full strength and multiple diluÂtions, and of the active ingredients. All disinfectants demAônstrated efficient virus inactivation. CA extern

Respiratory diseases negatively impact the global goat industry, but are understudied. There is a shortage of established and biological relevant in vitro or ex vivo assays to study caprine respiratory infections. Here, we describe the establishment of an in vitro system based on well-differentiated caprine airway epithelial cell (AEC) cultures grown under air liquid interface conditions as an experimental platform to study caprine respiratory pathogens. The functional differentiation of the AEC cultures was monitored and confirmed by light and immunofluorescence microscopy, scanning electron microscopy and examination of histological sections. We validated the functionality of the platform by studying Influenza D Virus (IDV) infection and Mycoplasma mycoides subsp. capri (Mmc) colonization over 5 days, including monitoring of infectious agents by titration and qPCR as well as colour changing units, respectively. The inoculation of caprine AEC cultures with IDV showed that efficient viral replication takes place, and revealed that IDV has a marked cell tropism for ciliated cells. Furthermore, AEC cultures were successfully infected with Mmc using a multiplicity of infection of 0.1 and colonization was monitored over several days. Altogether, these results demonstrate that our newly-established caprine AEC cultures can be used to investigate host-pathogen interactions of caprine respiratory pathogens.

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., Double membrane vesicles (DMV) are used as replication organelles by several RNA viruses. Applying proteomics and lipidomics, Tabata and Prasad et al. find that two cellular acyltransferases (AGPAT1/2), responsible for synthesis of phosphatidic acid, play a role in the DMV-biogenesis of HCV and SARS-CoV-2, highlighting a common biogenesis mechanism for evolutionary distant positive-strand RNA viruses.

Biotin-based proximity labeling circumvents major pitfalls of classical biochemical approaches to identify protein–protein interactions. It consists of enzyme-catalyzed biotin tags ubiquitously apposed on proteins located in close proximity of the labeling enzyme, followed by affinity purification and identification of biotinylated proteins by mass spectrometry. Here we outline the methods by which the molecular microenvironment of the coronavirus replicase/transcriptase complex (RTC), i.e., proteins located within a close perimeter of the RTC, can be determined by different proximity labeling approaches using BirAR118G (BioID), TurboID, and APEX2. These factors represent a molecular signature of coronavirus RTCs and likely contribute to the viral life cycle, thereby constituting attractive targets for the development of antiviral intervention strategies.

Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2, a new member of the genus Betacoronavirus, is a pandemic virus, which has caused numerous fatalities, particularly in the elderly and persons with underlying morbidities. At present, there are no approved vaccines nor antiviral therapies available. The detection and quantification of SARS-CoV-2-neutralizing antibodies plays a crucial role in the assessment of the immune status of convalescent COVID-19 patients, evaluation of recombinant therapeutic antibodies, and the evaluation of novel vaccines. To detect SARS-CoV-2-neutralizing antibodies, classically, a virus-neutralization test has to be performed at biosafety level 3, considerably limiting the general use of this test. In the present work, a biosafety level 1 pseudotype virus assay based on a propagation-incompetent vesicular stomatitis virus (VSV) has been used to determine the neutralizing antibody titers in convalescent COVID-19 patients. The neutralization titers in serum of two independently analyzed patient cohorts were available within 18 h and correlated well with those obtained with a classical SARS-CoV-2 neutralization test (Pearson correlation coefficients of r = 0.929 and r = 0.939, respectively). Most convalescent COVID-19 patients had only low titers of neutralizing antibodies (ND50 &lt, 320). The sera of convalescent COVID-19 patients also neutralized pseudotype virus displaying the SARS-CoV-1 spike protein on their surface, which is homologous to the SARS-CoV-2 spike protein. In summary, we report a robust virus-neutralization assay, which can be used at low biosafety level 1 to rapidly quantify SARS-CoV-2-neutralizing antibodies in convalescent COVID-19 patients and vaccinated individuals.

With currently over 4 million confirmed cases worldwide, including more than 300’000 deaths, the current Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic has a major impact on the economy and health care system. Currently, a limited amount of prophylactic or therapeutic intervention options are available against SARS-CoV-2. In this study, we screened 400 compounds from the antimicrobial ‘Pandemic Response Box’ library for inhibiting properties against SARS-CoV-2. We identified sixteen compounds that potently inhibited SARS-CoV-2 replication, of which five compounds displayed equal or even higher antiviral activity compared to Remdesivir. These results show that five compounds should be further investigated for their mode of action, safety and efficacy against SARS-CoV-2.Highlights400 compounds from the pandemic response box were tested for antiviral activity against SARS-CoV-2.5 compounds had an equal or higher antiviral efficacy towards SARS-CoV-2, compared to the nucleoside analogue Remdesivir.

The human conductive respiratory tract spans a long anatomical distance and represents an important barrier to constrain invading respiratory pathogens. The disparate ambient temperatures found in the upper and lower respiratory tract have been demonstrated to influence the replication kinetics of common cold viruses as well as the associated host responses. Here, we employed the human airway epithelial cell (hAEC) culture model to investigate the impact of ambient temperatures found in the upper and lower respiratory tract, 33°C and 37°C, respectively, on the viral replication kinetics and host innate immune response dynamics during SARS-CoV-2 and SARS-CoV infections. Strikingly, SARS-CoV-2, in contrast to SARS-CoV, replicated more efficiently at temperatures encountered in the upper respiratory tract, and displayed higher sensitivity to type I and type III IFNs than SARS-CoV. Time-resolved transcriptome analysis highlighted a temperature-dependent induction of IFN-mediated antiviral response, whose amplitude inversely correlated with the replication kinetic efficiencies of both SARS-CoV-2 and SARS-CoV at temperatures found in the upper and lower respiratory tract. Altogether, these data reflect clinical features of SARS-CoV-2 and SARS-CoV and subsequently, their associated human-to-human transmission efficiencies. They provide crucial insights of the profound impact of ambient temperatures on viral replication and associated pivotal virus - host interaction dynamics. This knowledge can be exploited for the development of novel intervention strategies against SARS-CoV-2.

Respiratory viruses, such as the 2009 pandemic strain of influenza A virus (IAV, H1N1pdm09), target cells found in the human respiratory epithelium. These cells, which form a pseudostratified epithelial layer along the airways, constitute the first line of defence against respiratory pathogens and play a crucial role in the host antiviral response. However, despite their key role in host defence, it remains unknown how distinct cell types in the respiratory epithelium respond to IAV infection and how these responses may contribute to IAV-induced pathogenesis and overall disease outcome. Here, we used single cell RNA-sequencing (scRNA-seq) to dissect the host response to IAV infection in its natural target cells. scRNA-seq was performed on human airway epithelial cell (hAEC) cultures infected with either wild-type pandemic IAV (WT) or with a mutant version of IAV (NS1R38A) that induced a robust innate immune response. We then characterized both the host and viral transcriptomes of more than 19,000 single cells across the 5 major cell types populating the human respiratory epithelium. For all cell types, we observed a wide spectrum of viral burden among single infected cells and a disparate host response between infected and bystander populations. Interestingly, we also identified multiple key differences in the host response to IAV among individual cell types, including high levels of pro-inflammatory cytokines and chemokines in secretory and basal cells and an important role for luminal cells in sensing and restricting incoming virus. Multiple infected cell types were shown to upregulate interferons (IFN), with type III IFNs clearly dominating the antiviral response. Transcriptional changes in genes related to cell differentiation, cell migration, and tissue repair were also identified. Strikingly, we also detected a shift in viral host cell tropism from non-ciliated cells to ciliated cells at later stages of infection and observed major changes in the cellular composition. Microscopic analysis of both WT and NS1R38A virus-infected hAECs at various stages of IAV infection revealed that the transcriptional changes we observed at 18 hpi were likely driving the downstream histopathological alterations in the airway epithelium. To our knowledge, this is the first study to provide a comprehensive analysis of the cell type-specific host antiviral response to a respiratory virus infection in its natural target cells – namely, the human respiratory epithelium.

Zoonotic coronaviruses (CoVs) are significant threats to global health, as exemplified by the recent emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)1. Host immune responses to CoV are complex and regulated in part through antiviral interferons. However, the interferon-stimulated gene products that inhibit CoV are not well characterized2. Here, we show that interferon-inducible lymphocyte antigen 6 complex, locus E (LY6E) potently restricts cellular infection by multiple CoVs, including SARS-CoV, SARS-CoV-2, and Middle East respiratory syndrome coronavirus (MERS-CoV). Mechanistic studies revealed that LY6E inhibits CoV entry into cells by interfering with spike protein-mediated membrane fusion. Importantly, mice lacking Ly6e in hematopoietic cells were highly susceptible to murine CoV infection. Exacerbated viral pathogenesis in Ly6e knockout mice was accompanied by loss of hepatic and splenic immune cells and reduction in global antiviral gene pathways. Accordingly, we found that Ly6e directly protects primary B cells and dendritic cells from murine CoV infection. Our results demonstrate that LY6E is a critical antiviral immune effector that controls CoV infection and pathogenesis. These findings advance our understanding of immune-mediated control of CoV in vitro and in vivo, knowledge that could help inform strategies to combat infection by emerging CoV.

Reverse genetics has been an indispensable tool revolutionising our insights into viral pathogenesis and vaccine development. Large RNA virus genomes, such as from Coronaviruses, are cumbersome to clone and to manipulate in E. coli hosts due to size and occasional instability1-3. Therefore, an alternative rapid and robust reverse genetics platform for RNA viruses would benefit the research community. Here we show the full functionality of a yeast-based synthetic genomics platform for the genetic reconstruction of diverse RNA viruses, including members of the Coronaviridae, Flaviviridae and Paramyxoviridae families. Viral subgenomic fragments were generated using viral isolates, cloned viral DNA, clinical samples, or synthetic DNA, and reassembled in one step in Saccharomyces cerevisiae using transformation associated recombination (TAR) cloning to maintain the genome as a yeast artificial chromosome (YAC). T7-RNA polymerase has been used to generate infectious RNA, which was then used to rescue viable virus. Based on this platform we have been able to engineer and resurrect chemically-synthetized clones of the recent epidemic SARS-CoV-24 in only a week after receipt of the synthetic DNA fragments. The technical advance we describe here allows to rapidly responding to emerging viruses as it enables the generation and functional characterization of evolving RNA virus variants - in real-time - during an outbreak.

Highlights • Hospital environment was contaminated when providing care to COVID-19 patients • Contaminated surfaces included COVID-19 patient care areas, hospital objects, and PPE • Hospital environment could be a source of virus spread among HCWs and patients • Contacting with contaminated surfaces may account for the early cases in HWCs, Objectives A large number of healthcare workers (HCWs) were infected by SARS-CoV-2 during the ongoing outbreak of COVID-19 in Wuhan, China. Hospitals are significant epicenters for the human-to-human transmission of the SARS-CoV-2 for HCWs, patients, and visitors. No data has been reported on the details of hospital environmental contamination status in the epicenter of Wuhan. Methods We collected 626 surface swabs within the Zhongnan Medical Center in Wuhan in the mist of the COVID-19 outbreak between February 7 - February 27, 2020. Dacron swabs were aseptically collected from the surfaces of 13 hospital function zones, five major objects, and three major PPE. The SARS-CoV-2 RNAs were detected by reverse transcription-PCR. Results The most contaminated zones were the intensive care unit specialized for taking care of novel coronavirus pneumonia (NCP) (31.9%), Obstetric Isolation Ward specialized for pregnant women with NCP (28.1%), and Isolation Ward for NCP (19.6%). We classified the 13 zones into four contamination levels. The most contaminated objects were self-service printers (20.0%), desktop/keyboard (16.8%), and doorknob (16.0%). Both hand sanitizer dispensers (20.3%) and gloves (15.4%) were the most contaminated PPE. Conclusion Our findings emphasize the urgent need to ensure adequate environmental cleaning, strengthen infection prevention training, and improve infection prevention among HCWs during the outbreak of COVID-19.

Biotin-based proximity labeling circumvents major pitfalls of classical biochemical approaches to identify protein-protein interactions. It consists of enzyme-catalyzed biotin tags ubiquitously apposed on proteins located in close proximity of the labeling enzyme, followed by affinity purification and identification of biotinylated proteins by mass spectrometry. Here we outline the methods by which the molecular microenvironment of the coronavirus replicase/transcriptase complex (RTC), i.e., proteins located within a close perimeter of the RTC, can be determined by different proximity labeling approaches using BirAR118G (BioID), TurboID, and APEX2. These factors represent a molecular signature of coronavirus RTCs and likely contribute to the viral life cycle, thereby constituting attractive targets for the development of antiviral intervention strategies.

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) depends on angiotensin converting enzyme 2 (ACE2) for cellular entry, but it might also rely on attachment receptors such as heparan sulfates. Several groups have recently demonstrated an affinity of the SARS-CoV2 spike protein for heparan sulfates and a reduced binding to cells in the presence of heparin or heparinase treatment. Here, we investigated the inhibitory activity of several sulfated and sulfonated molecules, which prevent interaction with heparan sulfates, against vesicular stomatitis virus (VSV)-pseudotyped-SARS-CoV-2 and the authentic SARS-CoV-2. Sulfonated cyclodextrins and nanoparticles that have recently shown broad-spectrum non-toxic virucidal activity against many heparan sulfates binding viruses showed inhibitory activity in the micromolar and nanomolar ranges, respectively. In stark contrast with the mechanisms that these compounds present for these other viruses, the inhibition against SARS-CoV-2 was found to be simply reversible.

Highlights • The anti-coronaviral compound K22 has broad antiviral activity against diverse nidoviruses. • K22 treatment reduces double-stranded RNA following nidovirus infection. • K22 displays antiviral activity in nidovirus infection models of different species., Recently, a novel antiviral compound (K22) that inhibits replication of a broad range of animal and human coronaviruses was reported to interfere with viral RNA synthesis by impairing double-membrane vesicle (DMV) formation (Lundin et al., 2014). Here we assessed potential antiviral activities of K22 against a range of viruses representing two (sub)families of the order Nidovirales, the Arteriviridae (porcine reproductive and respiratory syndrome virus [PRRSV], equine arteritis virus [EAV] and simian hemorrhagic fever virus [SHFV]), and the Torovirinae (equine torovirus [EToV] and White Bream virus [WBV]). Possible effects of K22 on nidovirus replication were studied in suitable cell lines. K22 concentrations significantly decreasing infectious titres of the viruses included in this study ranged from 25 to 50 μM. Reduction of double-stranded RNA intermediates of viral replication in nidovirus-infected cells treated with K22 confirmed the anti-viral potential of K22. Collectively, the data show that K22 has antiviral activity against diverse lineages of nidoviruses, suggesting that the inhibitor targets a critical and conserved step during nidovirus replication.

The virus family Flaviviridae encompasses several viruses, including (re)emerging viruses which cause widespread morbidity and mortality throughout the world. Members of this virus family are positive-strand RNA viruses and replicate their genome in close association with reorganized intracellular host cell membrane compartments., The virus family Flaviviridae encompasses several viruses, including (re)emerging viruses which cause widespread morbidity and mortality throughout the world. Members of this virus family are positive-strand RNA viruses and replicate their genome in close association with reorganized intracellular host cell membrane compartments. This evolutionarily conserved strategy facilitates efficient viral genome replication and contributes to evasion from host cell cytosolic defense mechanisms. We have previously described the identification of a small-compound inhibitor, K22, which exerts a potent antiviral activity against a broad range of coronaviruses by targeting membrane-bound viral RNA replication. To analyze the antiviral spectrum of this inhibitor, we assessed the inhibitory potential of K22 against several members of the Flaviviridae family, including the reemerging Zika virus (ZIKV). We show that ZIKV is strongly affected by K22. Time-of-addition experiments revealed that K22 acts during a postentry phase of the ZIKV life cycle, and combination regimens of K22 together with ribavirin (RBV) or interferon alpha (IFN-α) further increased the extent of viral inhibition. Ultrastructural electron microscopy studies revealed severe alterations of ZIKV-induced intracellular replication compartments upon infection of K22-treated cells. Importantly, the antiviral activity of K22 was demonstrated against several other members of the Flaviviridae family. It is tempting to speculate that K22 exerts its broad antiviral activity against several positive-strand RNA viruses via a similar mechanism and thereby represents an attractive candidate for development as a panviral inhibitor.

APOBEC3 family members are cytidine deaminases with roles in intrinsic responses to infection by retroviruses and retrotransposons, and in the control of other DNA viruses, such as herpesviruses, parvoviruses and hepatitis B virus. Although effects of APOBEC3 members on viral DNA have been demonstrated, it is not known whether they edit RNA genomes through cytidine deamination. Here, we investigated APOBEC3-mediated restriction of Coronaviridae. In experiments in vitro, three human APOBEC3 proteins (A3C, A3F and A3H) inhibited HCoV-NL63 infection and limited production of progeny virus, but did not cause hypermutation of the coronaviral genome. APOBEC3-mediated restriction was partially dependent on enzyme activity, and was reduced by the use of enzymatically inactive APOBEC3. Moreover, APOBEC3 proteins bound to the coronaviral nucleoprotein, and this interaction also affected viral replication. Although the precise molecular mechanism of deaminase-dependent inhibition of coronavirus replication remains elusive, our results further our understanding of APOBEC-mediated restriction of RNA virus infections.

Coronaviruses are of veterinary and medical importance and include highly pathogenic zoonotic viruses, such as SARS-CoV and MERS-CoV. They are known to efficiently evade early innate immune responses, manifesting in almost negligible expression of type-I interferons (IFN-I). This evasion strategy suggests an evolutionary conserved viral function that has evolved to prevent RNA-based sensing of infection in vertebrate hosts. Here we show that the coronavirus endonuclease (EndoU) activity is key to prevent early induction of double-stranded RNA (dsRNA) host cell responses. Replication of EndoU-deficient coronaviruses is greatly attenuated in vivo and severely restricted in primary cells even during the early phase of the infection. In macrophages we found immediate induction of IFN-I expression and RNase L-mediated breakdown of ribosomal RNA. Accordingly, EndoU-deficient viruses can retain replication only in cells that are deficient in IFN-I expression or sensing, and in cells lacking both RNase L and PKR. Collectively our results demonstrate that the coronavirus EndoU efficiently prevents simultaneous activation of host cell dsRNA sensors, such as Mda5, OAS and PKR. The localization of the EndoU activity at the site of viral RNA synthesis–within the replicase complex—suggests that coronaviruses have evolved a viral RNA decay pathway to evade early innate and intrinsic antiviral host cell responses., Author summary Coronaviruses are long known to be particularly successful in evading host innate immune responses. This manifests in barely detectable interferon responses during the first hours of infection and greatly facilitates establishment of robust virus replication. This phenotype of early innate immune evasion is common to all coronaviruses and can have detrimental consequences particular for infections with highly pathogenic coronaviruses, such as SARS-CoV and MERS-CoV. We therefore hypothesized that there must be an evolutionary conserved viral function that has evolved to prevent sensing of coronavirus infection by infected host cells. Our study now describes this function, namely the highly conserved coronavirus endoribonuclease activity. We found that coronaviruses that lack this enzymatic activity are readily visible to infected host cells that can now mount a swift and potent host response restricting virus replication within hours. Our study provides a new paradigm of a first layer of RNA virus innate immune evasion during the early phase of infection, that takes place at the site of RNA synthesis, and is based on removal of dsRNA that would otherwise trigger the simultaneous activation of cytoplasmic host cell sensors.

Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved intracellular catabolic transport route that generally allows the lysosomal degradation of cytoplasmic components, including bulk cytosol, protein aggregates, damaged or superfluous organelles and invading microbes. Target structures are sequestered by double-membrane vesicles called autophagosomes, which are formed through the concerted action of the autophagy (ATG)-related proteins. Until recently it was assumed that ATG proteins were exclusively involved in autophagy. A growing number of studies, however, have attributed functions to some of them that are distinct from their classical role in autophagosome biogenesis. Autophagy-independent roles of the ATG proteins include the maintenance of cellular homeostasis and resistance to pathogens. For example, they assist and enhance the turnover of dead cells and microbes upon their phagocytic engulfment, and inhibit murine norovirus replication. Moreover, bone resorption by osteoclasts, innate immune regulation triggered by cytoplasmic DNA and the ER-associated degradation regulation all have in common the requirement of a subset of ATG proteins. Microorganisms such as coronaviruses, Chlamydia trachomatis or Brucella abortus have even evolved ways to manipulate autophagy-independent functions of ATG proteins in order to ensure the completion of their intracellular life cycle. Taken together these novel mechanisms add to the repertoire of functions and extend the number of cellular processes involving the ATG proteins.

Highlights • Coronavirus replication is tied to formation of double-membrane organelles (DMOs). • DMO-making genes are conserved across the Nidovirales. • Here, we interpret recent experiments on the role and importance of coronavirus DMOs., The replication of coronaviruses, as in other positive-strand RNA viruses, is closely tied to the formation of membrane-bound replicative organelles inside infected cells. The proteins responsible for rearranging cellular membranes to form the organelles are conserved not just among the Coronaviridae family members, but across the order Nidovirales. Taken together, these observations suggest that the coronavirus replicative organelle plays an important role in viral replication, perhaps facilitating the production or protection of viral RNA. However, the exact nature of this role, and the specific contexts under which it is important have not been fully elucidated. Here, we collect and interpret the recent experimental evidence about the role and importance of membrane-bound organelles in coronavirus replication.

The transport pathway of autophagy is characterized by double‐membrane vesicles called autophagosomes, which are formed through the concerted action of the conserved ATG. Until recently it was assumed that ATG proteins were exclusively involved in autophagy. A growing number of studies, however, have revealed that specific ATG proteins have roles beyond their involvement in autophagosome biogenesis. This article reviews this fast expanding new field of the autophagy research., Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved intracellular catabolic transport route that generally allows the lysosomal degradation of cytoplasmic components, including bulk cytosol, protein aggregates, damaged or superfluous organelles and invading microbes. Target structures are sequestered by double‐membrane vesicles called autophagosomes, which are formed through the concerted action of the autophagy (ATG)‐related proteins. Until recently it was assumed that ATG proteins were exclusively involved in autophagy. A growing number of studies, however, have attributed functions to some of them that are distinct from their classical role in autophagosome biogenesis. Autophagy‐independent roles of the ATG proteins include the maintenance of cellular homeostasis and resistance to pathogens. For example, they assist and enhance the turnover of dead cells and microbes upon their phagocytic engulfment, and inhibit murine norovirus replication. Moreover, bone resorption by osteoclasts, innate immune regulation triggered by cytoplasmic DNA and the ER‐associated degradation regulation all have in common the requirement of a subset of ATG proteins. Microorganisms such as coronaviruses, Chlamydia trachomatis or Brucella abortus have even evolved ways to manipulate autophagy‐independent functions of ATG proteins in order to ensure the completion of their intracellular life cycle. Taken together these novel mechanisms add to the repertoire of functions and extend the number of cellular processes involving the ATG proteins.

Positive-sense RNA viruses hijack intracellular membranes that provide niches for viral RNA synthesis and a platform for interactions with host proteins. However, little is known about host factors at the interface between replicase complexes and the host cytoplasm. We engineered a biotin ligase into a coronaviral replication/transcription complex (RTC) and identified >500 host proteins constituting the RTC microenvironment. siRNA-silencing of each RTC-proximal host factor demonstrated importance of vesicular trafficking pathways, ubiquitin-dependent and autophagy-related processes, and translation initiation factors. Notably, detection of translation initiation factors at the RTC was instrumental to visualize and demonstrate active translation proximal to replication complexes of several coronaviruses. Collectively, we establish a spatial link between viral RNA synthesis and diverse host factors of unprecedented breadth. Our data may serve as a paradigm for other positive-strand RNA viruses and provide a starting point for a comprehensive analysis of critical virus-host interactions that represent targets for therapeutic intervention., eLife digest Coronaviruses can infect the nose and throat and are a main cause of the common cold. Infections are usually mild and short-lived, but sometimes they can turn nasty. In 2002 and 2012, two dangerous new coronaviruses emerged and caused diseases known as SARS and MERS. These viruses caused much more serious symptoms and in some cases proved deadly. The question is, why are some coronaviruses more dangerous than others? Scientists know that the body's response to virus infection can make a difference to whether someone had mild or severe disease. So, to understand why some coronaviruses cause a cold and others kill, they also need to learn how people react to virus infection. Coronaviruses hijack membranes inside cells and turn them into virus factories. Within these factories, the viruses build molecular machinery called replicase complexes to copy their genetic code, which is needed for the next generation of virus particles. The viruses steal and repurpose proteins from their host cell that will assist in the copying process. However, scientists do not yet know which host proteins are essential for the virus to multiply. So, to find out, V’kovski et al. developed a way to tag any host protein that came near the virus factories. The new technique involved attaching an enzyme called a biotin ligase to the replicase complex. This enzyme acts as a molecular label gun, attaching a chemical tag to any protein that comes within ten nanometres. The label gun revealed that more than 500 different proteins come into contact with the replicase complex. To find out what these proteins were doing, the next step was to switch off their genes one by one. This revealed the key cell machinery that coronaviruses hijack when they are replicating. It included the cell's cargo transport system, the waste disposal system, and the protein production system. Using these systems allows the viruses to copy their genetic code next to machines that can turn it straight into viral proteins. These new results provide clues about which proteins viruses actually need from their host cells. They also do not just apply to coronaviruses. Other viruses use similar strategies to complete their infection cycle. These findings could help researchers to understand more generally about how viruses multiply. In the future, this knowledge could lead to new ways to combat virus infections.