Attenuation of SARS‐CoV‐2 replication and associated inflammation by concomitant targeting of viral and host cap 2'‐O‐ribose methyltransferases

The SARS‐CoV‐2 infection cycle is a multistage process that relies on functional interactions between the host and the pathogen. Here, we repurposed antiviral drugs against both viral and host enzymes to pharmaceutically block methylation of the viral RNA 2''‐O‐ribose cap needed for viral immune escape. We find that the host cap 2''‐O‐ribose methyltransferase MTr1 can compensate for loss of viral NSP16 methyltransferase in facilitating virus replication. Concomitant inhibition of MTr1 and NSP16 efficiently suppresses SARS‐CoV‐2 replication. Using in silico target‐based drug screening, we identify a bispecific MTr1/NSP16 inhibitor with anti‐SARS‐CoV‐2 activity in vitro and in vivo but with unfavorable side effects. We further show antiviral activity of inhibitors that target independent stages of the host SAM cycle providing the methyltransferase co‐substrate. In particular, the adenosylhomocysteinase (AHCY) inhibitor DZNep is antiviral in in vitro, in ex vivo, and in a mouse infection model and synergizes with existing COVID‐19 treatments. Moreover, DZNep exhibits a strong immunomodulatory effect curbing infection‐induced hyperinflammation and reduces lung fibrosis markers ex vivo. Thus, multispecific and metabolic MTase inhibitors constitute yet unexplored treatment options against COVID‐19.     

DDX60 selectively reduces translation off viral type II internal ribosome entry sites

Co‐opting host cell protein synthesis is a hallmark of many virus infections. In response, certain host defense proteins limit mRNA translation globally, albeit at the cost of the host cell''s own protein synthesis. Here, we describe an interferon‐stimulated helicase, DDX60, that decreases translation from viral internal ribosome entry sites (IRESs). DDX60 acts selectively on type II IRESs of encephalomyocarditis virus (EMCV) and foot and mouth disease virus (FMDV), but not by other IRES types or by 5′ cap. Correspondingly, DDX60 reduces EMCV and FMDV (type II IRES) replication, but not that of poliovirus or bovine enterovirus 1 (BEV‐1; type I IRES). Furthermore, replacing the IRES of poliovirus with a type II IRES is sufficient for DDX60 to inhibit viral replication. Finally, DDX60 selectively modulates the amount of translating ribosomes on viral and in vitro transcribed type II IRES mRNAs, but not 5′ capped mRNA. Our study identifies a novel facet in the repertoire of interferon‐stimulated effector genes, the selective downregulation of translation from viral type II IRES elements.     

Mechanisms of phosphatidylserine influence on viral production: A computational model of Ebola virus matrix protein assembly

Ebola virus (EBOV) infections continue to pose a global public health threat, with high mortality rates and sporadic outbreaks in Central and Western Africa. A quantitative understanding of the key processes driving EBOV assembly and budding could provide valuable insights to inform drug development. Here, we use a computational model to evaluate EBOV matrix assembly. Our model focuses on the assembly kinetics of VP40, the matrix protein in EBOV, and its interaction with phosphatidylserine (PS) in the host cell membrane. It has been shown that mammalian cells transfected with VP40-expressing plasmids are capable of producing virus-like particles (VLPs) that closely resemble EBOV virions. Previous studies have also shown that PS levels in the host cell membrane affects VP40 association with the plasma membrane inner leaflet and that lower membrane PS levels result in lower VLP production. Our computational findings indicate that PS may also have a direct influence on VP40 VLP assembly and budding, where a higher PS level will result in a higher VLP budding rate and filament dissociation rate. Our results further suggest that the assembly of VP40 filaments follow the nucleation-elongation theory, where initialization and oligomerization of VP40 are two distinct steps in the assembly process. Our findings advance the current understanding of VP40 VLP formation by identifying new possible mechanisms of PS influence on VP40 assembly. We propose that these mechanisms could inform treatment strategies targeting PS alone or in combination with other VP40 assembly steps.     

Microtubules are not required to generate a nascent axon in embryonic spinal neurons in vivo

Our understanding of the cell behaviours and cytoskeletal requirements of axon formation is largely derived from in vitro models but how these relate to axon formation in vivo is not clear. In vitro, neurons progress through a well‐defined multineurite stage to form an axon and both actin and microtubules cooperate to drive the first steps in neurite and axon morphogenesis. However, these steps are not recapitulated in vivo, and it is not clear whether the underlying cell biological mechanisms may differ also. Here, we investigate the mechanisms that regulate axon formation in embryonic zebrafish spinal neurons in vivo. We find microtubule organising centres are located distant from the site of axon initiation, and microtubule plus‐ends are not enriched in the axon during axon initiation. Focal F‐actin accumulation precedes axon formation, and we find that nocodazole‐treated neurons with no detectable microtubules are still able to form nascent axonal protrusions that are approximately 10‐μm long, dilated and relatively long‐lived. We suggest spinal axon formation in vivo is fundamentally different from axon formation in in vitro models.     

LincRNA‐EPS impairs host antiviral immunity by antagonizing viral RNA–PKR interaction

LincRNA‐EPS is an important regulator in inflammation. However, the role of lincRNA‐EPS in the host response against viral infection is unexplored. Here, we show that lincRNA‐EPS is downregulated in macrophages infected with different viruses including VSV, SeV, and HSV‐1. Overexpression of lincRNA‐EPS facilitates viral infection, while deficiency of lincRNA‐EPS protects the host against viral infection in vitro and in vivo. LincRNA‐EPS ^−/− macrophages show elevated expression of antiviral interferon‐stimulated genes (ISGs) such as Mx1, Oas2, and Ifit2 at both basal and inducible levels. However, IFN‐β, the key upstream inducer of these ISGs, is downregulated in lincRNA‐EPS ^−/− macrophages compared with control cells. RNA pulldown and mass spectrometry results indicate that lincRNA‐EPS binds to PKR and antagonizes the viral RNA–PKR interaction. PKR activates STAT1 and induces antiviral ISGs independent of IFN‐I induction. LincRNA‐EPS inhibits PKR‐STAT1‐ISGs signaling and thus facilitates viral infection. Our study outlines an alternative antiviral pathway, with downregulation of lincRNA‐EPS promoting the induction of PKR‐STAT1‐dependent ISGs, and reveals a potential therapeutic target for viral infectious diseases.     

Chemogenetic profiling reveals PP2A‐independent cytotoxicity of proposed PP2A activators iHAP1 and DT‐061

Protein phosphatase 2A (PP2A) is an abundant phosphoprotein phosphatase that acts as a tumor suppressor. For this reason, compounds able to activate PP2A are attractive anticancer agents. The compounds iHAP1 and DT‐061 have recently been reported to selectively stabilize specific PP2A‐B56 complexes to mediate cell killing. We were unable to detect direct effects of iHAP1 and DT‐061 on PP2A‐B56 activity in biochemical assays and composition of holoenzymes. Therefore, we undertook genome‐wide CRISPR‐Cas9 synthetic lethality screens to uncover biological pathways affected by these compounds. We found that knockout of mitotic regulators is synthetic lethal with iHAP1 while knockout of endoplasmic reticulum (ER) and Golgi components is synthetic lethal with DT‐061. Indeed we showed that iHAP1 directly blocks microtubule assembly both in vitro and in vivo and thus acts as a microtubule poison. In contrast, DT‐061 disrupts both the Golgi apparatus and the ER and lipid synthesis associated with these structures. Our work provides insight into the biological pathways perturbed by iHAP1 and DT‐061 causing cellular toxicity and argues that these compounds cannot be used for dissecting PP2A‐B56 biology.     

Mammalian adipogenesis regulator (Areg) cells use retinoic acid signalling to be non‐ and anti‐adipogenic in age‐dependent manner

Adipose stem and precursor cells (ASPCs) give rise to adipocytes and determine the composition and plasticity of adipose tissue. Recently, several studies have demonstrated that ASPCs partition into at least three distinct cell subpopulations, including the enigmatic CD142⁺ cells. An outstanding challenge is to functionally characterise this population, as discrepant properties, from adipogenic to non‐ and anti‐adipogenic, have been reported for these cells. To resolve these phenotypic ambiguities, we characterised mammalian subcutaneous CD142⁺ ASPCs across various experimental conditions, demonstrating that CD142⁺ ASPCs exhibit high molecular and phenotypic robustness. Specifically, we find these cells to be firmly non‐ and anti‐adipogenic both in vitro and in vivo, with their inhibitory signals also impacting adipogenic human cells. However, these CD142⁺ ASPC‐specific properties exhibit surprising temporal phenotypic alterations, and emerge only in an age‐dependent manner. Finally, using multi‐omic and functional assays, we show that the inhibitory nature of these adipogenesis‐regulatory CD142⁺ ASPCs (Aregs) is driven by specifically expressed secretory factors that cooperate with the retinoic acid signalling pathway to transform the adipogenic state of CD142⁻ ASPCs into a non‐adipogenic, Areg‐like state.     

SARS‐CoV‐2 nucleocapsid suppresses host pyroptosis by blocking Gasdermin D cleavage

SARS‐CoV‐2 is an emerging coronavirus that causes dysfunctions in multiple human cells and tissues. Studies have looked at the entry of SARS‐CoV‐2 into host cells mediated by the viral spike protein and human receptor ACE2. However, less is known about the cellular immune responses triggered by SARS‐CoV‐2 viral proteins. Here, we show that the nucleocapsid of SARS‐CoV‐2 inhibits host pyroptosis by blocking Gasdermin D (GSDMD) cleavage. SARS‐CoV‐2‐infected monocytes show enhanced cellular interleukin‐1β (IL‐1β) expression, but reduced IL‐1β secretion. While SARS‐CoV‐2 infection promotes activation of the NLRP3 inflammasome and caspase‐1, GSDMD cleavage and pyroptosis are inhibited in infected human monocytes. SARS‐CoV‐2 nucleocapsid protein associates with GSDMD in cells and inhibits GSDMD cleavage in vitro and in vivo. The nucleocapsid binds the GSDMD linker region and hinders GSDMD processing by caspase‐1. These insights into how SARS‐CoV‐2 antagonizes cellular inflammatory responses may open new avenues for treating COVID‐19 in the future.     

ZAKβ is activated by cellular compression and mediates contraction‐induced MAP kinase signaling in skeletal muscle

Mechanical inputs give rise to p38 and JNK activation, which mediate adaptive physiological responses in various tissues. In skeletal muscle, contraction‐induced p38 and JNK signaling ensure adaptation to exercise, muscle repair, and hypertrophy. However, the mechanisms by which muscle fibers sense mechanical load to activate this signaling have remained elusive. Here, we show that the upstream MAP3K ZAKβ is activated by cellular compression induced by osmotic shock and cyclic compression in vitro, and muscle contraction in vivo. This function relies on ZAKβ''s ability to recognize stress fibers in cells and Z‐discs in muscle fibers when mechanically perturbed. Consequently, ZAK‐deficient mice present with skeletal muscle defects characterized by fibers with centralized nuclei and progressive adaptation towards a slower myosin profile. Our results highlight how cells in general respond to mechanical compressive load and how mechanical forces generated during muscle contraction are translated into MAP kinase signaling.     

A plant reovirus hijacks the DNAJB12–Hsc70 chaperone complex to promote viral spread in its planthopper vector

Many viruses usurp the functions of endoplasmic reticulum (ER) for virus‐encoded membrane proteins proper functional folding or assembly to promote virus spread. Southern rice black‐streaked dwarf virus (SRBSDV), a plant reovirus, exploits virus‐containing tubules composed of nonstructural membrane protein P7‐1 to spread in its planthopper vector Sogatella furcifera. Here, we report that two factors of the ER‐associated degradation (ERAD) machinery, the ER chaperone DNAJB12 and its cytosolic co‐chaperone Hsc70, are activated by SRBSDV to facilitate ER‐to‐cytosol export of P7‐1 tubules in S. furcifera. Both P7‐1 of SRBSDV and Hsc70 directly bind to the J‐domain of DNAJB12. DNAJB12 overexpression induces ER retention of P7‐1, but Hsc70 overexpression promotes the transport of P7‐1 from the ER to the cytosol to initiate tubule assembly. Thus, P7‐1 is initially retained in the ER by interaction with DNAJB12 and then delivered to Hsc70. Furthermore, the inhibitors of the ATPase activity of Hsc70 reduce P7‐1 tubule assembly, suggesting that the proper folding and assembly of P7‐1 tubules is dependent on the ATPase activity of Hsc70. The DNAJB12–Hsc70 chaperone complex is recruited to P7‐1 tubules in virus‐infected midgut epithelial cells in S. furcifera. The knockdown of DNAJB12 or Hsc70 strongly inhibits P7‐1 tubule assembly in vivo, finally suppressing effective viral spread in S. furcifera. Taken together, our results indicate that the DNAJB12–Hsc70 chaperone complex in the ERAD machinery facilitates the ER‐to‐cytosol transport of P7‐1 for proper assembly of tubules, enabling viral spread in insect vectors in a manner dependent on ATPase activity of Hsc70.     

F‐actin dynamics in midgut cells enables virus persistence in vector insects

Hemipteran insects that transmit plant viruses in a persistent circulative manner acquire, retain and transmit viruses for their entire life. The mechanism enabling this persistence has remained unclear for many years. Here, we determined how wheat dwarf virus (WDV) persists in its leafhopper vector Psammotettix alienus. We found that WDV caused the up‐regulation of actin‐depolymerizing factor (ADF) at the mRNA and protein levels in the midgut cells of leafhoppers after experiencing a WDV acquisition access period (AAP) of 6, 12 or 24 h. Experimental inhibition of F‐actin depolymerization by jasplakinolide and dsRNA injection led to lower virus accumulation levels and transmission efficiencies, suggesting that depolymerization of F‐actin regulated by ADF is essential for WDV invasion of midgut cells. Exogenous viral capsid protein (CP) inhibited ADF depolymerization of actin filaments in vitro and in Spodoptera frugiperda 9 (Sf9) cells because the CP competed with actin to bind ADF and then blocked actin filament disassembly. Interestingly, virions colocalized with ADF after a 24‐h AAP, just as actin polymerization occurred, indicating that the binding of CP with ADF affects the ability of ADF to depolymerize F‐actin, inhibiting WDV entry. Similarly, the luteovirus barley yellow dwarf virus also induced F‐actin depolymerization and then polymerization in the gut cells of its vector Schizaphis graminum. Thus, F‐actin dynamics are altered by nonpropagative viruses in midgut cells to enable virus persistence in vector insects.     

Nonproductive exposure of PBMCs to SARS‐CoV‐2 induces cell‐intrinsic innate immune responses

Cell‐intrinsic responses mounted in PBMCs during mild and severe COVID‐19 differ quantitatively and qualitatively. Whether they are triggered by signals emitted by productively infected cells of the respiratory tract or result from physical interaction with virus particles remains unclear. Here, we analyzed susceptibility and expression profiles of PBMCs from healthy donors upon ex vivo exposure to SARS‐CoV and SARS‐CoV‐2. In line with the absence of detectable ACE2 receptor expression, human PBMCs were refractory to productive infection. RT–PCR experiments and single‐cell RNA sequencing revealed JAK/STAT‐dependent induction of interferon‐stimulated genes (ISGs) but not proinflammatory cytokines. This SARS‐CoV‐2‐specific response was most pronounced in monocytes. SARS‐CoV‐2‐RNA‐positive monocytes displayed a lower ISG signature as compared to bystander cells of the identical culture. This suggests a preferential invasion of cells with a low ISG baseline profile or delivery of a SARS‐CoV‐2‐specific sensing antagonist upon efficient particle internalization. Together, nonproductive physical interaction of PBMCs with SARS‐CoV‐2‐ and, to a much lesser extent, SARS‐CoV particles stimulate JAK/STAT‐dependent, monocyte‐accentuated innate immune responses that resemble those detected in vivo in patients with mild COVID‐19.     

Inhibition of Marburg Virus Budding by Nonneutralizing Antibodies to the Envelope Glycoprotein

The envelope glycoprotein (GP) of Marburg virus (MARV) and Ebola virus (EBOV) is responsible for virus entry into host cells and is known as the only target of neutralizing antibodies. While knowledge about EBOV-neutralizing antibodies and the mechanism for the neutralization of infectivity is being accumulated gradually, little is known about antibodies that can efficiently regulate MARV infectivity. Here we show that MARV GP-specific monoclonal antibodies AGP127-8 (IgG1) and MGP72-17 (IgM), which do not inhibit the GP-mediated entry of MARV into host cells, drastically reduced the budding and release of progeny viruses from infected cells. These antibodies similarly inhibited the formation of virus-like particles (VLPs) consisting of GP, the viral matrix protein, and nucleoprotein, whereas the Fab fragment of AGP127-8 showed no inhibitory effect. Morphological analyses revealed that filamentous VLPs were bunched on the surface of VLP-producing cells cultured in the presence of the antibodies. These results demonstrate a novel mechanism of the antibody-mediated inhibition of MARV budding, in which antibodies arrest unformed virus particles on the cell surface. Our data lead to the idea that such antibodies, like classical neutralizing antibodies, contribute to protective immunity against MARV and that the “classical” neutralizing activity is not the only indicator of a protective antibody that may be available for prophylactic and therapeutic use.     

Cathepsin B & L Are Not Required for Ebola Virus Replication

Ebola virus (EBOV), family Filoviridae, emerged in 1976 on the African continent. Since then it caused several outbreaks of viral hemorrhagic fever in humans with case fatality rates up to 90% and remains a serious Public Health concern and biothreat pathogen. The most pathogenic and best-studied species is Zaire ebolavirus (ZEBOV). EBOV encodes one viral surface glycoprotein (GP), which is essential for replication, a determinant of pathogenicity and an important immunogen. GP mediates viral entry through interaction with cellular surface molecules, which results in the uptake of virus particles via macropinocytosis. Later in this pathway endosomal acidification activates the cysteine proteases Cathepsin B and L (CatB, CatL), which have been shown to cleave ZEBOV-GP leading to subsequent exposure of the putative receptor-binding and fusion domain and productive infection. We studied the effect of CatB and CatL on in vitro and in vivo replication of EBOV. Similar to previous findings, our results show an effect of CatB, but not CatL, on ZEBOV entry into cultured cells. Interestingly, cell entry by other EBOV species (Bundibugyo, Côte d''Ivoire, Reston and Sudan ebolavirus) was independent of CatB or CatL as was EBOV replication in general. To investigate whether CatB and CatL have a role in vivo during infection, we utilized the mouse model for ZEBOV. Wild-type (control), catB^−/− and catL^−/− mice were equally susceptible to lethal challenge with mouse-adapted ZEBOV with no difference in virus replication and time to death. In conclusion, our results show that CatB and CatL activity is not required for EBOV replication. Furthermore, EBOV glycoprotein cleavage seems to be mediated by an array of proteases making targeted therapeutic approaches difficult.     

The Ebola virus soluble glycoprotein contributes to viral pathogenesis by activating the MAP kinase signaling pathway

Ebola virus (EBOV) expresses three different glycoproteins (GPs) from its GP gene. The primary product, soluble GP (sGP), is secreted in abundance during infection. EBOV sGP has been discussed as a potential pathogenicity factor, however, little is known regarding its functional role. Here, we analyzed the role of sGP in vitro and in vivo. We show that EBOV sGP has two different functions that contribute to infectivity in tissue culture. EBOV sGP increases the uptake of virus particles into late endosomes in HEK293 cells, and it activates the mitogen-activated protein kinase (MAPK) signaling pathway leading to increased viral replication in Huh7 cells. Furthermore, we analyzed the role of EBOV sGP on pathogenicity using a well-established mouse model. We found an sGP-dependent significant titer increase of EBOV in the liver of infected animals. These results provide new mechanistic insights into EBOV pathogenicity and highlight EBOV sGP as a possible therapeutic target.     

The nucleoporin Nup50 activates the Ran guanine nucleotide exchange factor RCC1 to promote NPC assembly at the end of mitosis

During mitotic exit, thousands of nuclear pore complexes (NPCs) assemble concomitant with the nuclear envelope to build a transport‐competent nucleus. Here, we show that Nup50 plays a crucial role in NPC assembly independent of its well‐established function in nuclear transport. RNAi‐mediated downregulation in cells or immunodepletion of Nup50 protein in Xenopus egg extracts interferes with NPC assembly. We define a conserved central region of 46 residues in Nup50 that is crucial for Nup153 and MEL28/ELYS binding, and for NPC interaction. Surprisingly, neither NPC interaction nor binding of Nup50 to importin α/β, the GTPase Ran, or chromatin is crucial for its function in the assembly process. Instead, an N‐terminal fragment of Nup50 can stimulate the Ran GTPase guanine nucleotide exchange factor RCC1 and NPC assembly, indicating that Nup50 acts via the Ran system in NPC reformation at the end of mitosis. In support of this conclusion, Nup50 mutants defective in RCC1 binding and stimulation cannot replace the wild‐type protein in in vitro NPC assembly assays, whereas excess RCC1 can compensate the loss of Nup50.     

Zinc pyrithione is a potent inhibitor of PLPro and cathepsin L enzymes with ex vivo inhibition of SARS-CoV-2 entry and replication

Zinc pyrithione (1a), together with its analogues 1b–h and ruthenium pyrithione complex 2a, were synthesised and evaluated for the stability in biologically relevant media and anti-SARS-CoV-2 activity. Zinc pyrithione revealed potent in vitro inhibition of cathepsin L (IC₅₀=1.88 ± 0.49 µM) and PL^Pro (IC₅₀=0.50 ± 0.07 µM), enzymes involved in SARS-CoV-2 entry and replication, respectively, as well as antiviral entry and replication properties in an ex vivo system derived from primary human lung tissue. Zinc complexes 1b–h expressed comparable in vitro inhibition. On the contrary, ruthenium complex 2a and the ligand pyrithione a itself expressed poor inhibition in mentioned assays, indicating the importance of the selection of metal core and structure of metal complex for antiviral activity. Safe, effective, and preferably oral at-home therapeutics for COVID-19 are needed and as such zinc pyrithione, which is also commercially available, could be considered as a potential therapeutic agent against SARS-CoV-2.     

An engineered bacterial therapeutic lowers urinary oxalate in preclinical models and in silico simulations of enteric hyperoxaluria

Enteric hyperoxaluria (EH) is a metabolic disease caused by excessive absorption of dietary oxalate leading to the formation of chronic kidney stones and kidney failure. There are no approved pharmaceutical treatments for EH. SYNB8802 is an engineered bacterial therapeutic designed to consume oxalate in the gut and lower urinary oxalate as a potential treatment for EH. Oral administration of SYNB8802 leads to significantly decreased urinary oxalate excretion in healthy mice and non‐human primates, demonstrating the strain''s ability to consume oxalate in vivo. A mathematical modeling framework was constructed that combines in vitro and in vivo preclinical data to predict the effects of SYNB8802 administration on urinary oxalate excretion in humans. Simulations of SYNB8802 administration predict a clinically meaningful lowering of urinary oxalate excretion in healthy volunteers and EH patients. Together, these findings suggest that SYNB8802 is a promising treatment for EH.