Endoplasmic reticulum-associated SARS-CoV-2 ORF3a elicits heightened cytopathic effects despite robust ER-associated degradation.

IMPORTANCE: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has tragically claimed millions of lives through coronavirus disease 2019 (COVID-19), and there remains a critical gap in our understanding of the precise molecular mechanisms responsible for the associated fatality. One key viral factor of interest is the SARS-CoV-2 ORF3a protein, which has been identified as a potent inducer of host cellular proinflammatory responses capable of triggering the catastrophic cytokine storm, a primary contributor to COVID-19-related deaths. Moreover, ORF3a, much like the spike protein, exhibits a propensity for frequent mutations, with certain variants linked to the severity of COVID-19. Our previous research unveiled two distinct types of ORF3a mutant proteins, categorized by their subcellular localizations, setting the stage for a comparative investigation into the functional and mechanistic disparities between these two types of ORF3a variants. Given the clinical significance and functional implications of the natural ORF3a mutations, the findings of this study promise to provide invaluable insights into the potential roles undertaken by these mutant ORF3a proteins in the pathogenesis of COVID-19.

Palmitoylation of the hemagglutinin of influenza B virus by ER-localized DHHC enzymes 1, 2, 4, and 6 is required for efficient virus replication.

IMPORTANCE: Influenza viruses are a public health concern since they cause seasonal outbreaks and occasionally pandemics. Our study investigates the importance of a protein modification called "palmitoylation" in the replication of influenza B virus. Palmitoylation involves attaching fatty acids to the viral protein hemagglutinin and has previously been studied for influenza A virus. We found that this modification is important for the influenza B virus to replicate, as mutating the sites where palmitate is attached prevented the virus from generating viable particles. Our experiments also showed that this modification occurs in the endoplasmic reticulum. We identified the specific enzymes responsible for this modification, which are different from those involved in palmitoylation of HA of influenza A virus. Overall, our research illuminates the similarities and differences in fatty acid attachment to HA of influenza A and B viruses and identifies the responsible enzymes, which might be promising targets for anti-viral therapy.

SARS-CoV-2 protein ORF8 limits expression levels of Spike antigen and facilitates immune evasion of infected host cells.

Recovery from COVID-19 depends on the ability of the host to effectively neutralize virions and infected cells, a process largely driven by antibody-mediated immunity. However, with the newly emerging variants that evade Spike-targeting antibodies, re-infections and breakthrough infections are increasingly common. A full characterization of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mechanisms counteracting antibody-mediated immunity is therefore needed. Here, we report that ORF8 is a virally encoded SARS-CoV-2 factor that controls cellular Spike antigen levels. We show that ORF8 limits the availability of mature Spike by inhibiting host protein synthesis and retaining Spike at the endoplasmic reticulum, reducing cell-surface Spike levels and recognition by anti-SARS-CoV-2 antibodies. In conditions of limited Spike availability, we found ORF8 restricts Spike incorporation during viral assembly, reducing Spike levels in virions. Cell entry of these virions then leaves fewer Spike molecules at the cell surface, limiting antibody recognition of infected cells. Based on these findings, we propose that SARS-CoV-2 variants may adopt an ORF8-dependent strategy that facilitates immune evasion of infected cells for extended viral production.

Reticulons promote formation of ER-derived double-membrane vesicles that facilitate SARS-CoV-2 replication.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the etiologic agent for the global COVID-19 pandemic, triggers the formation of endoplasmic reticulum (ER)-derived replication organelles, including double-membrane vesicles (DMVs), in the host cell to support viral replication. Here, we clarify how SARS-CoV-2 hijacks host factors to construct the DMVs. We show that the ER morphogenic proteins reticulon-3 (RTN3) and RTN4 help drive DMV formation, enabling viral replication, which leads to productive infection. Different SARS-CoV-2 variants, including the delta variant, use the RTN-dependent pathway to promote infection. Mechanistically, our results reveal that the membrane-embedded reticulon homology domain (RHD) of the RTNs is sufficient to functionally support viral replication and physically engage NSP3 and NSP4, two viral non-structural membrane proteins known to induce DMV formation. Our findings thus identify the ER morphogenic RTN3 and RTN4 membrane proteins as host factors that help promote the biogenesis of SARS-CoV-2-induced DMVs, which can act as viral replication platforms.

The role of NSP6 in the biogenesis of the SARS-CoV-2 replication organelle.

SARS-CoV-2, like other coronaviruses, builds a membrane-bound replication organelle to enable RNA replication1. The SARS-CoV-2 replication organelle is composed of double-membrane vesicles (DMVs) that are tethered to the endoplasmic reticulum (ER) by thin membrane connectors2, but the viral proteins and the host factors involved remain unknown. Here we identify the viral non-structural proteins (NSPs) that generate the SARS-CoV-2 replication organelle. NSP3 and NSP4 generate the DMVs, whereas NSP6, through oligomerization and an amphipathic helix, zippers ER membranes and establishes the connectors. The NSP6(ΔSGF) mutant, which arose independently in the Alpha, Beta, Gamma, Eta, Iota and Lambda variants of SARS-CoV-2, behaves as a gain-of-function mutant with a higher ER-zippering activity. We identified three main roles for NSP6: first, to act as a filter in communication between the replication organelle and the ER, by allowing lipid flow but restricting the access of ER luminal proteins to the DMVs; second, to position and organize DMV clusters; and third, to mediate contact with lipid droplets (LDs) through the LD-tethering complex DFCP1-RAB18. NSP6 thus acts as an organizer of DMV clusters and can provide a selective means of refurbishing them with LD-derived lipids. Notably, both properly formed NSP6 connectors and LDs are required for the replication of SARS-CoV-2. Our findings provide insight into the biological activity of NSP6 of SARS-CoV-2 and of other coronaviruses, and have the potential to fuel the search for broad antiviral agents.

Rhinovirus C replication is associated with the endoplasmic reticulum and triggers cytopathic effects in an in vitro model of human airway epithelium.

The clinical impact of rhinovirus C (RV-C) is well-documented; yet, the viral life cycle remains poorly defined. Thus, we characterized RV-C15 replication at the single-cell level and its impact on the human airway epithelium (HAE) using a physiologically-relevant in vitro model. RV-C15 replication was restricted to ciliated cells where viral RNA levels peaked at 12 hours post-infection (hpi), correlating with elevated titers in the apical compartment at 24hpi. Notably, infection was associated with a loss of polarized expression of the RV-C receptor, cadherin-related family member 3. Visualization of double-stranded RNA (dsRNA) during RV-C15 replication revealed two distinct replication complex arrangements within the cell, likely corresponding to different time points in infection. To further define RV-C15 replication sites, we analyzed the expression and colocalization of giantin, phosphatidylinositol-4-phosphate, and calnexin with dsRNA. Despite observing Golgi fragmentation by immunofluorescence during RV-C15 infection as previously reported for other RVs, a high ratio of calnexin-dsRNA colocalization implicated the endoplasmic reticulum as the primary site for RV-C15 replication in HAE. RV-C15 infection was also associated with elevated stimulator of interferon genes (STING) expression and the induction of incomplete autophagy, a mechanism used by other RVs to facilitate non-lytic release of progeny virions. Notably, genetic depletion of STING in HAE attenuated RV-C15 and -A16 (but not -B14) replication, corroborating a previously proposed proviral role for STING in some RV infections. Finally, RV-C15 infection resulted in a temporary loss in epithelial barrier integrity and the translocation of tight junction proteins while a reduction in mucociliary clearance indicated cytopathic effects on epithelial function. Together, our findings identify both shared and unique features of RV-C replication compared to related rhinoviruses and define the impact of RV-C on both epithelial cell organization and tissue functionality-aspects of infection that may contribute to pathogenesis in vivo.

Gain-of-function genetic screening identifies the antiviral function of TMEM120A via STING activation.

Zika virus (ZIKV) infection can be associated with neurological pathologies, such as microcephaly in newborns and Guillain-Barre syndrome in adults. Effective therapeutics are currently not available. As such, a comprehensive understanding of virus-host interactions may guide the development of medications for ZIKV. Here we report a human genome-wide overexpression screen to identify host factors that regulate ZIKV infection and find TMEM120A as a ZIKV restriction factor. TMEM120A overexpression significantly inhibits ZIKV replication, while TMEM120A knockdown increases ZIKV infection in cell lines. Moreover, Tmem120a knockout in mice facilitates ZIKV infection in primary mouse embryonic fibroblasts (MEF) cells. Mechanistically, the antiviral activity of TMEM120A is dependent on STING, as TMEM120A interacts with STING, promotes the translocation of STING from the endoplasmic reticulum (ER) to ER-Golgi intermediate compartment (ERGIC) and enhances the phosphorylation of downstream TBK1 and IRF3, resulting in the expression of multiple antiviral cytokines and interferon-stimulated genes. In summary, our gain-of-function screening identifies TMEM120A as a key activator of the antiviral signaling of STING.

Harnessing autophagy to fight SARS-CoV-2: An update in view of recent drug development efforts.

Drug repurposing is an attractive option for identifying new treatment strategies, in particular in extraordinary situations of urgent need such as the current coronavirus disease 2019 (Covid-19) pandemic. Recently, the World Health Organization announced testing of three drugs as potential Covid-19 therapeutics that are known for their dampening effect on the immune system. Thus, the underlying concept of selecting these drugs is to temper the potentially life-threatening overshooting of the immune system reacting to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. This viewpoint discusses the possibility that the impact of these and other drugs on autophagy contributes to their therapeutic effect by hampering the SARS-CoV-2 life cycle.

NSP9 of SARS-CoV-2 attenuates nuclear transport by hampering nucleoporin 62 dynamics and functions in host cells.

Nuclear pore complexes (NPC) regulate molecular traffics on nuclear envelope, which plays crucial roles during cell fate specification and diseases. The viral accessory protein NSP9 of SARS-CoV-2 is reported to interact with nucleoporin 62 (NUP62), a structural component of the NPC, but its biological impact on the host cell remain obscure. Here, we established new cell line models with ectopic NSP9 expression and determined the subcellular destination and biological functions of NSP9. Confocal imaging identified NSP9 to be largely localized in close proximity to the endoplasmic reticulum. In agreement with the subcellular distribution of NSP9, association of NSP9 with NUP62 was observed in cytoplasm. Furthermore, the overexpression of NSP9 correlated with a reduction of NUP62 expression on the nuclear envelope, suggesting that attenuating NUP62 expression might have contributed to defective NPC formation. Importantly, the loss of NUP62 impaired translocation of p65, a subunit of NF-κB, upon TNF-α stimulation. Concordantly, NSP9 over-expression blocked p65 nuclear transport. Taken together, these data shed light on the molecular mechanisms underlying the modulation of host cells during SARS-CoV-2 infection.

Lunapark-dependent formation of a virus-induced ER exit site contains multi-tubular ER junctions that promote viral ER-to-cytosol escape.

Viruses rearrange host membranes to support different entry steps. Polyomavirus simian virus 40 (SV40) reorganizes the endoplasmic reticulum (ER) membrane to generate focus structures that enable virus ER-to-cytosol escape, a decisive infection step. The molecular architecture of the ER exit site that might illuminate why it is ideally suited for membrane penetration is unknown. Here 3D focused ion beam scanning electron microscopy (FIB-SEM) reconstruction reveals that the ER focus structure consists of multi-tubular ER junctions where SV40 preferentially localizes, suggesting that tubular branch points are virus ER-to-cytosol penetration sites. Functional analysis demonstrates that lunapark-an ER membrane protein that typically stabilizes three-way ER junctions-relocates to the ER foci, where it supports focus formation, leading to SV40 ER escape and infection. Our results reveal how a virus repurposes the activity of an ER membrane protein to form a virus-induced ER substructure required for membrane escape and suggest that ER tubular junctions are vulnerable sites exploited by viruses for membrane penetration.

How Viruses Hijack and Modify the Secretory Transport Pathway.

Eukaryotic cells contain dynamic membrane-bound organelles that are constantly remodeled in response to physiological and environmental cues. Key organelles are the endoplasmic reticulum, the Golgi apparatus and the plasma membrane, which are interconnected by vesicular traffic through the secretory transport route. Numerous viruses, especially enveloped viruses, use and modify compartments of the secretory pathway to promote their replication, assembly and cell egression by hijacking the host cell machinery. In some cases, the subversion mechanism has been uncovered. In this review, we summarize our current understanding of how the secretory pathway is subverted and exploited by viruses belonging to Picornaviridae, Coronaviridae, Flaviviridae,Poxviridae, Parvoviridae and Herpesviridae families.

The Biogenesis of Dengue Virus Replication Organelles Requires the ATPase Activity of Valosin-Containing Protein.

The dengue virus (DENV) causes the most prevalent arthropod-borne viral disease worldwide. While its incidence is increasing in many countries, there is no approved antiviral therapy currently available. In infected cells, the DENV induces extensive morphological alterations of the endoplasmic reticulum (ER) to generate viral replication organelles (vRO), which include convoluted membranes (CM) and vesicle packets (VP) hosting viral RNA replication. The viral non-structural protein NS4B localizes to vROs and is absolutely required for viral replication through poorly defined mechanisms, which might involve cellular protein partners. Previous interactomic studies identified the ATPase valosin-containing protein (VCP) as a DENV NS4B-interacting host factor in infected cells. Using both pharmacological and dominant-negative inhibition approaches, we show, in this study, that VCP ATPase activity is required for efficient DENV replication. VCP associates with NS4B when expressed in the absence of other viral proteins while in infected cells, both proteins colocalize within large DENV-induced cytoplasmic structures previously demonstrated to be CMs. Consistently, VCP inhibition dramatically reduces the abundance of DENV CMs in infected cells. Most importantly, using a recently reported replication-independent plasmid-based vRO induction system, we show that de novo VP biogenesis is dependent on VCP ATPase activity. Overall, our data demonstrate that VCP ATPase activity is required for vRO morphogenesis and/or stability. Considering that VCP was shown to be required for the replication of other flaviviruses, our results argue that VCP is a pan-flaviviral host dependency factor. Given that new generation VCP-targeting drugs are currently evaluated in clinical trials for cancer treatment, VCP may constitute an attractive broad-spectrum antiviral target in drug repurposing approaches.

Disruption of the Golgi Apparatus and Contribution of the Endoplasmic Reticulum to the SARS-CoV-2 Replication Complex.

A variety of immunolabeling procedures for both light and electron microscopy were used to examine the cellular origins of the host membranes supporting the SARS-CoV-2 replication complex. The endoplasmic reticulum has long been implicated as a source of membrane for the coronavirus replication organelle. Using dsRNA as a marker for sites of viral RNA synthesis, we provide additional evidence supporting ER as a prominent source of membrane. In addition, we observed a rapid fragmentation of the Golgi apparatus which is visible by 6 h and complete by 12 h post-infection. Golgi derived lipid appears to be incorporated into the replication organelle although protein markers are dispersed throughout the infected cell. The mechanism of Golgi disruption is undefined, but chemical disruption of the Golgi apparatus by brefeldin A is inhibitory to viral replication. A search for an individual SARS-CoV-2 protein responsible for this activity identified at least five viral proteins, M, S, E, Orf6, and nsp3, that induced Golgi fragmentation when expressed in eukaryotic cells. Each of these proteins, as well as nsp4, also caused visible changes to ER structure as shown by correlative light and electron microscopy (CLEM). Collectively, these results imply that specific disruption of the Golgi apparatus is a critical component of coronavirus replication.

Dengue and Zika virus capsid proteins bind to membranes and self-assemble into liquid droplets with nucleic acids.

Dengue virus (DENV) and Zika virus (ZIKV) capsid proteins efficiently recruit and surround the viral RNA at the endoplasmic reticulum (ER) membrane to yield nascent viral particles. However, little is known either about the molecular mechanisms by which multiple copies of capsid proteins assemble into nucleocapsids (NCs) or how the NC is recruited and wrapped by the ER membrane during particle morphogenesis. Here, we measured relevant interactions concerning this viral process using purified DENV and ZIKV capsid proteins, membranes mimicking the ER lipid composition, and nucleic acids in in vitro conditions to understand the biophysical properties of the RNA genome encapsidation process. We found that both ZIKV and DENV capsid proteins bound to liposomes at liquid-disordered phase regions, docked exogenous membranes, and RNA molecules. Liquid-liquid phase separation is prone to occur when positively charged proteins interact with nucleic acids, which is indeed the case for the studied capsids. We characterized these liquid condensates by measuring nucleic acid partition constants and the extent of water dipolar relaxation, observing a cooperative process for the formation of the new phase that involves a distinct water organization. Our data support a new model in which capsid-RNA complexes directly bind the ER membrane, seeding the process of RNA recruitment for viral particle assembly. These results contribute to our understanding of the viral NC formation as a stable liquid-liquid phase transition, which could be relevant for dengue and Zika gemmation, opening new avenues for antiviral intervention.

Increased susceptibility of human endothelial cells to infections by SARS-CoV-2 variants.

Coronavirus disease 2019 (COVID-19) spawned a global health crisis in late 2019 and is caused by the novel coronavirus SARS-CoV-2. SARS-CoV-2 infection can lead to elevated markers of endothelial dysfunction associated with higher risk of mortality. It is unclear whether endothelial dysfunction is caused by direct infection of endothelial cells or is mainly secondary to inflammation. Here, we investigate whether different types of endothelial cells are susceptible to SARS-CoV-2. Human endothelial cells from different vascular beds including umbilical vein endothelial cells, coronary artery endothelial cells (HCAEC), cardiac and lung microvascular endothelial cells, or pulmonary arterial cells were inoculated in vitro with SARS-CoV-2. Viral spike protein was only detected in HCAECs after SARS-CoV-2 infection but not in the other endothelial cells tested. Consistently, only HCAEC expressed the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2), required for virus infection. Infection with the SARS-CoV-2 variants B.1.1.7, B.1.351, and P.2 resulted in significantly higher levels of viral spike protein. Despite this, no intracellular double-stranded viral RNA was detected and the supernatant did not contain infectious virus. Analysis of the cellular distribution of the spike protein revealed that it co-localized with endosomal calnexin. SARS-CoV-2 infection did induce the ER stress gene EDEM1, which is responsible for clearance of misfolded proteins from the ER. Whereas the wild type of SARS-CoV-2 did not induce cytotoxic or pro-inflammatory effects, the variant B.1.1.7 reduced the HCAEC cell number. Of the different tested endothelial cells, HCAECs showed highest viral uptake but did not promote virus replication. Effects on cell number were only observed after infection with the variant B.1.1.7, suggesting that endothelial protection may be particularly important in patients infected with this variant.

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.

Roles of the Endogenous Lunapark Protein during Flavivirus Replication.

The endoplasmic reticulum (ER) of eukaryotic cells is a dynamic organelle, which undergoes continuous remodeling. At the three-way tubular junctions of the ER, the lunapark (LNP) protein acts as a membrane remodeling factor to stabilize these highly curved membrane junctions. In addition, during flavivirus infection, the ER membrane is invaginated to form vesicles (Ve) for virus replication. Thus, LNP may have roles in the generation or maintenance of the Ve during flavivirus infection. In this study, our aim was to characterize the functions of LNP during flavivirus infection and investigate the underlying mechanisms of these functions. To specifically study virus replication, we generated cell lines expressing replicons of West Nile virus (Kunjin strain) or Langat virus. By using these replicon platforms and electron microscopy, we showed that depletion of LNP resulted in reduced virus replication, which is due to its role in the generation of the Ve. By using biochemical assays and high-resolution microscopy, we found that LNP is recruited to the Ve and the protein interacts with the nonstructural protein (NS) 4B. Therefore, these data shed new light on the interactions between flavivirus and host factors during viral replication.

How DNA and RNA Viruses Exploit Host Chaperones to Promote Infection.

To initiate infection, a virus enters a host cell typically via receptor-dependent endocytosis. It then penetrates a subcellular membrane, reaching a destination that supports transcription, translation, and replication of the viral genome. These steps lead to assembly and morphogenesis of the new viral progeny. The mature virus finally exits the host cell to begin the next infection cycle. Strikingly, viruses hijack host molecular chaperones to accomplish these distinct entry steps. Here we highlight how DNA viruses, including polyomavirus and the human papillomavirus, exploit soluble and membrane-associated chaperones to enter a cell, penetrating and escaping an intracellular membrane en route for infection. We also describe the mechanism by which RNA viruses-including flavivirus and coronavirus-co-opt cytosolic and organelle-selective chaperones to promote viral endocytosis, protein biosynthesis, replication, and assembly. These examples underscore the importance of host chaperones during virus infection, potentially revealing novel antiviral strategies to combat virus-induced diseases.

Fatty Acid Synthase Is Involved in Classical Swine Fever Virus Replication by Interaction with NS4B.

Classical swine fever virus (CSFV), a member of the genus Pestivirus of the family Flaviviridae, relies on host machinery to complete its life cycle. Previous studies have shown a close connection between virus infection and fatty acid biosynthesis, mainly regulated by fatty acid synthase (FASN). However, the molecular action of how FASN participates in CSFV replication remains to be elucidated. In this study, two chemical inhibitors of the fatty acid synthesis pathway [5-(tetradecyloxy)-2-furoic acid (TOFA) and tetrahydro-4-methylene-2R-octyl-5-oxo-3S-furancarboxylic acid (C75)] significantly impaired the late stage of viral propagation, suggesting CSFV replication required fatty acid synthesis. We next found that CSFV infection stimulated the expression of FASN, whereas knockdown of FASN inhibited CSFV replication. Furthermore, confocal microscopy showed that FASN participated in the formation of replication complex (RC), which was associated with the endoplasmic reticulum (ER). Interestingly, CSFV NS4B interacted with FASN and promoted overexpression of FASN, which is regulated by functional Rab18. Moreover, we found that FASN regulated the formation of lipid droplets (LDs) upon CSFV infection, promoting virus proliferation. Taken together, our work provides mechanistic insight into the role of FASN in the viral life of CSFV, and it highlights the potential antiviral target for the development of therapeutics against pestiviruses. IMPORTANCE Classical swine fever, caused by classical swine fever virus (CSFV), is one of the notifiable diseases by the World Organization for Animal Health (OIE) and causes significant financial losses to the pig industry globally. CSFV, like other (+)-strand RNA viruses, requires lipid and sterol biosynthesis for efficient replication. However, the role of lipid metabolism in CSFV replication remains unknown. Here, we found that fatty acid synthase (FASN) was involved in viral propagation. Moreover, FASN is recruited to CSFV replication sites in the endoplasmic reticulum (ER) and interacts with NS4B to regulate CSFV replication that requires Rab18. Furthermore, we speculated that lipid droplet (LD) biosynthesis, indirectly regulated by FASN, ultimately promotes CSFV replication. Our results highlight a critical role for de novo fatty acid synthesis in CSFV infection, which might help control this devastating virus.

TMEM41B is a host factor required for the replication of diverse coronaviruses including SARS-CoV-2.

Antiviral therapeutics are a front-line defense against virally induced diseases. Because viruses frequently mutate to escape direct inhibition of viral proteins, there is interest in targeting the host proteins that the virus must co-opt to complete its replication cycle. However, a detailed understanding of the interactions between the virus and the host cell is necessary in order to facilitate development of host-directed therapeutics. As a first step, we performed a genome-wide loss of function screen using the alphacoronavirus HCoV-229E to better define the interactions between coronaviruses and host factors. We report the identification and validation of an ER-resident host protein, TMEM41B, as an essential host factor for not only HCoV-229E but also genetically distinct coronaviruses including the pandemic betacoronavirus SARS-CoV-2. We show that the protein is required at an early, but post-receptor engagement, stage of the viral lifecycle. Further, mechanistic studies revealed that although the protein was not enriched at replication complexes, it likely contributes to viral replication complex formation via mobilization of cholesterol and other lipids to facilitate host membrane expansion and curvature. Continued study of TMEM41B and the development of approaches to prevent its function may lead to broad spectrum anti-coronavirus therapeutics.