Reverse genetics systems for SARS‐CoV‐2 Wang, Wenhao; Peng, Xiaoxue; Jin, Yunyun ...
Journal of medical virology,
July 2022, Letnik:
94, Številka:
7
Journal Article
Recenzirano
Odprti dostop
The ongoing pandemic of coronavirus disease 2019 (COVID‐19) has caused severe public health crises and heavy economic losses. Limited knowledge about this deadly virus impairs our capacity to set up ...a toolkit against it. Thus, more studies on severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) biology are urgently needed. Reverse genetics systems, including viral infectious clones and replicons, are powerful platforms for viral research projects, spanning many aspects such as the rescues of wild‐type or mutant viral particles, the investigation of viral replication mechanism, the characterization of viral protein functions, and the studies on viral pathogenesis and antiviral drug development. The operations on viral infectious clones are strictly limited in the Biosafety Level 3 (BSL3) facilities, which are insufficient, especially during the pandemic. In contrast, the operation on the noninfectious replicon can be performed in Biosafety Level 2 (BSL2) facilities, which are widely available. After the outbreak of COVID‐19, many reverse genetics systems for SARS‐CoV‐2, including infectious clones and replicons are developed and given plenty of options for researchers to pick up according to the requirement of their research works. In this review, we summarize the available reverse genetics systems for SARS‐CoV‐2, by highlighting the features of these systems, and provide a quick guide for researchers, especially those without ample experience in operating viral reverse genetics systems.
Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is a poor inducer of innate antiviral immunity, and the underlying mechanism still needs further investigation. Here, we reported that ...SARS‐CoV‐2 NSP7 inhibited the production of type I and III interferons (IFNs) by targeting the RIG‐I/MDA5, Toll‐like receptor (TLR3)‐TRIF, and cGAS‐STING signaling pathways. SARS‐CoV‐2 NSP7 suppressed the expression of IFNs and IFN‐stimulated genes induced by poly (I:C) transfection and infection with Sendai virus or SARS‐CoV‐2 virus‐like particles. NSP7 impaired type I and III IFN production activated by components of the cytosolic dsRNA‐sensing pathway, including RIG‐I, MDA5, and MAVS, but not TBK1, IKKε, and IRF3‐5D, an active form of IRF3. In addition, NSP7 also suppressed TRIF‐ and STING‐induced IFN responses. Mechanistically, NSP7 associated with RIG‐I and MDA5 prevented the formation of the RIG‐I/MDA5−MAVS signalosome and interacted with TRIF and STING to inhibit TRIF‐TBK1 and STING‐TBK1 complex formation, thus reducing the subsequent IRF3 phosphorylation and nuclear translocation that are essential for IFN induction. In addition, ectopic expression of NSP7 impeded innate immune activation and facilitated virus replication. Taken together, SARS‐CoV‐2 NSP7 dampens type I and III IFN responses via disruption of the signal transduction of the RIG‐I/MDA5−MAVS, TLR3‐TRIF, and cGAS‐STING signaling pathways, thus providing novel insights into the interactions between SARS‐CoV‐2 and innate antiviral immunity.
The protein activator of protein kinase R (PKR) (PACT) has been shown to play a crucial role in stimulating the host antiviral response through the activation of PKR, retinoic acid‐inducible gene I, ...and melanoma differentiation‐associated protein 5. Whether PACT can inhibit viral replication independent of known mechanisms is still unrevealed. In this study, we show that, like many viruses, severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) hijacks GSK‐3β to facilitate its replication. GSK‐3β‐induced phosphorylation on N protein increased the interaction between N protein and nsp3. Thus, GSK‐3β‐N‐nsp3 cascade promotes viral replication. Although SARS‐CoV‐2 can sabotage the activation of AKT, the upstream proteins suppressing the activation of GSK‐3β, we found that the host can use PACT, another protein kinase, instead of AKT to decrease the activity of GSK‐3β and the interaction between PACT and GSK‐3β is enhanced upon viral infection. Moreover, PACT inhibited the activity of GSK‐3β independent of its well‐studied double‐stranded RNA binding and PKR activating ability. In summary, this study identified an unknown function of PACT in inhibiting SARS‐CoV‐2 replication through the blockage of GSK‐3β‐N‐nsp3 cascade.
We herein develop two β-galactosidase (β-Gal) activatable NIR fluorescent probes for visualizing ovarian cancers. Particularly, probe BOD-M-βGal produced NIR-II emission light at 900-1300 nm upon ...β-Gal activation. By using our activatable and target specific NIR-II probe for deep-tissue imaging of β-Gal overexpressed ovarian cancer cells, rapid and accurate imaging of ovarian tumors in nude mice was achieved.
A β-galactosidase (β-Gal) activatable NIR-II fluorescent probe for visualizing ovarian cancers.
c-Myc is known to promote glutamine usage by upregulating glutaminase (GLS), which converts glutamine to glutamate that is catabolized in the TCA cycle. Here we report that in a number of human and ...murine cells and cancers, Myc induces elevated expression of glutamate-ammonia ligase (GLUL), also termed glutamine synthetase (GS), which catalyzes the de novo synthesis of glutamine from glutamate and ammonia. This is through upregulation of a Myc transcriptional target thymine DNA glycosylase (TDG), which promotes active demethylation of the GS promoter and its increased expression. Elevated expression of GS promotes cell survival under glutamine limitation, while silencing of GS decreases cell proliferation and xenograft tumor growth. Upon GS overexpression, increased glutamine enhances nucleotide synthesis and amino acid transport. These results demonstrate an unexpected role of Myc in inducing glutamine synthesis and suggest a molecular connection between DNA demethylation and glutamine metabolism in Myc-driven cancers.
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•Myc induces expression of glutamine synthetase (GS)•Increased GS expression is mediated by TDG-mediated promoter demethylation•GS promotes biosynthesis of asparagine and ribonucleotides, and amino acid transport•Suppression of GS decreases cell survival, proliferation, and oncogenesis
Bott et al. reveal that oncogenic Myc induces active DNA demethylation to increase glutamine synthetase (GS) expression and boost glutamine-dependent anabolic processes, including nucleotide synthesis and amino acid transport. GS modulates cell sensitivity to glutamine shortage, as well as proliferation and tumorigenesis.
As one of the most rapidly evolving proteins of the genus Betacoronavirus, open reading frames (ORF8's) function and potential pathological consequence in vivo are still obscure. In this study, we ...show that the secretion of ORF8 is dependent on its N‐terminal signal peptide sequence and can be inhibited by reactive oxygen species scavenger and endoplasmic reticulum‐Golgi transportation inhibitor in cultured cells. To trace the effect of its possible in vivo secretion, we examined the plasma samples of coronavirus disease 2019 (COVID‐19) convalescent patients and found that the patients aged from 40 to 60 had higher antibody titers than those under 40. To explore ORF8's in vivo function, we administered the mice with ORF8 via tail‐vein injection to simulate the circulating ORF8 in the patient. Although no apparent difference in body weight, food intake, and vitality was detected between vehicle‐ and ORF8‐treated mice, the latter displayed morphological abnormalities of testes and epididymides, as indicated by the loss of the central ductal lumen accompanied by a decreased fertility in 5‐week‐old male mice. Furthermore, the analysis of gene expression in the testes between vehicle‐ and ORF8‐treated mice identified a decreased expression of Col1a1, the loss of which is known to be associated with mice's infertility. Although whether our observation in mice could be translated to humans remains unclear, our study provides a potential mouse model that can be used to investigate the impact of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection on the human reproductive system.
Doxorubicin, an anthracycline chemotherapeutic agent, is widely used in the treatment of many cancers. However, doxorubicin posts a great risk of adverse cardiovascular events, which are thought to ...be caused by oxidative stress. We recently reported that the ubiquitin E3 ligase TRIM21 interacts and ubiquitylates p62 and negatively regulates the p62-Keap1-Nrf2 antioxidant pathway. Therefore, we sought to determine the role TRIM21 in cardiotoxicity induced by oxidative damage.
Using TRIM21 knockout mice, we examined the effects of TRIM21 on cardiotoxicity induced by two oxidative damage models: the doxorubicin treatment model and the Left Anterior Descending (LAD) model. We also explored the underlying mechanism by RNA-sequencing of the heart tissues, and by treating the mouse embryonic fibroblasts (MEFs), immortalized rat cardiomyocyte line H9c2, and immortalized human cardiomyocyte line AC16 with doxorubicin.
TRIM21 knockout mice are protected from heart failure and fatality in both the doxorubicin and LAD models. Hearts of doxorubicin-treated wild-type mice exhibit deformed mitochondria and elevated level of lipid peroxidation reminiscent of ferroptosis, which is alleviated in TRIM21 knockout hearts. Mechanistically, TRIM21-deficient heart tissues and cultured MEFs and H9c2 cells display enhanced p62 sequestration of Keap1 and are protected from doxorubicin-induced ferroptosis. Reconstitution of wild-type but not the E3 ligase-dead and the p62 binding-deficient TRIM21 mutants impedes the protection from doxorubicin-induced cell death.
Our study demonstrates that TRIM21 ablation protects doxorubicin-induced cardiotoxicity and illustrates a new function of TRIM21 in ferroptosis, and suggests TRIM21 as a therapeutic target for reducing chemotherapy-related cardiotoxicity.
NIH (CA129536; DK108989): data collection, analysis.
Shanghai Pujiang Program (19PJ1401900): data collection.
National Natural Science Foundation (31971161): data collection.
Department of Veteran Affairs (BX004083): data collection.
Tianjin Science and Technology Plan Project (17ZXMFSY00020): data collection.
The SARS-CoV-2 3-chymotrypsin-like protease (3CLpro or Mpro) is a key cysteine protease for viral replication and transcription, making it an attractive target for antiviral therapies to combat the ...COVID-19 disease. Here, we demonstrate that bismuth drug colloidal bismuth subcitrate (CBS) is a potent inhibitor for 3CLpro
in vitro
and
in cellulo
. Rather than targeting the cysteine residue at the catalytic site, CBS binds to an allosteric site and results in dissociation of the 3CLpro dimer and proteolytic dysfunction. Our work provides direct evidence that CBS is an allosteric inhibitor of SARS-CoV-2 3CLpro.
Colloidal bismuth subcitrate (CBS) is an allosteric inhibitor of 3-chymotrypsin-like protease (3CLpro) in SARS-CoV-2. CBS binding causes dimeric 3CLpro dissociation and proteolytic dysfunction, leading to the suppression of SARS-CoV-2 replication.
Abstract
Some supernovae, such as pair-instability supernovae, are predicted to have a duration of more than a year in the observer frame. To constrain the rates of supernovae lasting for more than a ...year, we conducted a long-term deep transient survey using Hyper Suprime-Cam (HSC) on the 8.2 m Subaru telescope. HSC is a wide-field (a 1.75 deg
2
field-of-view) camera and it can efficiently conduct transient surveys. We observed the same 1.75 deg
2
field repeatedly using the
g-
,
r-
,
i-
, and
z-
band filters with the typical depth of 26 mag for four seasons (from late 2016 to early 2020). Using these data, we searched for transients lasting for more than a year. Two supernovae were detected in two continuous seasons, one supernova was detected in three continuous seasons, but no transients lasted for all four seasons searched. The discovery rate of supernovae lasting for more than a year with the typical limiting magnitudes of 26 mag is constrained to be
. All the long-lasting supernovae we found are likely Type IIn supernovae and our results indicate that about 40% of Type IIn supernovae have long-lasting light curves. No plausible pair-instability supernova candidates lasting for more than a year are discovered. By comparing the survey results and survey simulations, we constrain the luminous pair-instability supernova rate up to
z
≃ 3 is of the order of 100 Gpc
−3
yr
−1
at most, which is 0.01–0.1% of the core-collapse supernova rate.
Glutamine is thought to play an important role in cancer cells by being deaminated via glutaminolysis to α-ketoglutarate (aKG) to fuel the tricarboxylic acid (TCA) cycle. Supporting this notion, aKG ...supplementation can restore growth/survival of glutamine-deprived cells. However, pancreatic cancers are often poorly vascularized and limited in glutamine supply, in alignment with recent concerns on the significance of glutaminolysis in pancreatic cancer. Here, we show that aKG-mediated rescue of glutamine-deprived pancreatic ductal carcinoma (PDAC) cells requires glutamate ammonia ligase (GLUL), the enzyme responsible for de novo glutamine synthesis. GLUL-deficient PDAC cells are capable of the TCA cycle but defective in aKG-coupled glutamine biosynthesis and subsequent nitrogen anabolic processes. Importantly, GLUL expression is elevated in pancreatic cancer patient samples and in mouse PDAC models. GLUL ablation suppresses the development of KrasG12D-driven murine PDAC. Therefore, GLUL-mediated glutamine biosynthesis couples the TCA cycle with nitrogen anabolism and plays a critical role in PDAC.
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•GLUL expression is high in human pancreatic cancers and in mouse PDAC models•aKG rescue of glutamine-deprived cells requires GLUL•De novo glutamine synthesis is essential for nitrogen anabolic processes•Genetic ablation of GLUL suppresses mouse PDAC development
Bott et al. demonstrate that GLUL-mediated glutamine synthesis plays a critical role in converging the TCA cycle and nitrogen metabolism to promote nitrogen-dependent anabolic processes in pancreatic cancer. Ablation of GLUL suppresses PDAC development and may have important clinical implications.
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