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Post by Admin on Nov 6, 2021 21:22:19 GMT
The number of new COVID-19 infections in Japan dropped to 229 on Sunday-which is less than 1 percent of the 25,900 active cases on Aug 20. According to research carried out by some Japanese scientists, a genome of the Delta variant, an enzyme called nsp14, has mutated resulting in reduced activity or inability to reproduce or mutate further-which can be called a self-detonation or self-destruction phenomenon. That has prompted some researchers to speculate that SARS-CoV-2 might be "weakening". SARS-CoV-2 is among those viruses that can mutate easily, sometimes within a week. The Delta variant of the virus, which is highly infectious, has already mutated further into 40 more variants, some of which are highly infectious and more harmful. As with most other viruses, the more the novel coronavirus mutates, the weaker the new variants are likely to become, and the nsp14 enzyme seems to be hastening this phenomenon. The nsp 14 enzyme was found in the majority of cases during the fifth wave of infections in Japan, supporting the hypothesis that SARS-CoV-2 might be weakening due to its own RNA variations. However, before we start celebrating, it is important to remember that only the variants with nsp14 enzyme might be weakening. Which means other variants could mutate further and become more infectious and harmful. Therefore, it is important for media outlets to report all sides of the story so that people are not misled. The Japanese research should not lead to undue speculation that the "novel coronavirus is dying". - Beijing News
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Post by Admin on Nov 7, 2021 4:33:27 GMT
When invaded by a virus, our body cells launch an alert to neighboring cells to increase their antiviral defenses to prevent the infection from spreading. Some viruses, however, manage to bypass this system by mimicking the host's RNA, preventing them from being detected by the infected cell and avoiding this alert. In the case of SARS-CoV-2, this mimicking uses a protein known as nsp14. This protein is also very important for virus multiplication, a task which is facilitated by its binding to the nsp10 protein, resulting in a protein complex. Interfering with nsp14 binding and with the nsp10-nsp14 protein complex is the aim of the most recent ITQB NOVA research in COVID-19, led by researchers Margarida Saramago, Rute Matos and Cecília Arraiano. The researchers began by performing the biochemical characterization of the nsp10-nsp14 protein complex, a known therapeutic target. "For the first time, it was possible to identify the amino acids which must be targeted in order to silence this complex", explains Rute Matos. The silencing of nsp14 also "makes it easier for the organism to identify the virus' messenger RNA and to activate the immune system before it replicates", adds fellow researcher Margarida Saramago. The discovery was only possible due to the collaboration between researchers from an experimental RNA laboratory and scientists from the bioinformatics area, who worked together to characterize the proteins. The construction of the three-dimensional model of the nsp14-nsp10 complex was based on the equivalent SARS-Cov-1 proteins. "It's like doing a facial composite", says bioinformatician Caio Souza, who built the model. With a very clear notion of the shape of the protein, it was possible to predict the most important amino acids. "We have a very detailed map of the target we must attack with future therapies," points out Diana Lousa, fellow bioinformatician and co-author of the study. "By silencing this protein, we will be able to "domesticate" a severe disease and turn it into a cold," explains Cecilia Arraiano, leader of one of the two laboratories involved. "It's as if we turned a wolf into a dog." This knowledge can now be used to develop antivirals, research which is now being carried out by the team. "Even with the hope of vaccines, it is essential to identify therapies capable of treating the infections that will continue to occur," explains Cláudio M. Soares, Director of ITQB NOVA and co-author of the work. "This type of research must be funded by public institutions," he adds. The scientific paper has been accepted for publication by the Federation of European Biological Societies (FEBS) in "The FEBS Journal". Reference: Saramago M, Bárria C, Costa VG, et al. New targets for drug design: Importance of nsp14/nsp10 complex formation for the 3’-5’ exoribonucleolytic activity on SARS-CoV-2. FEBS J. 2021. doi: 10.1111/febs.15815This article has been republished from the following materials. Note: material may have been edited for length and content. For further information, please contact the cited source.
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Post by Admin on Nov 8, 2021 4:07:25 GMT
Translational shutdown and evasion of the innate immune response by SARS-CoV-2 NSP14 protein PNAS June 15, 2021 118 (24) e2101161118; doi.org/10.1073/pnas.2101161118Contributed by Peter Cresswell, April 28, 2021 (sent for review January 19, 2021; reviewed by Mariano Garcia-Blanco and Stacy M. Horner) Significance To establish infection, pathogenic viruses have to overcome the type I interferon (IFN-I) antiviral response. A previous study demonstrated that the SARS-CoV-2 NSP14 is able to inhibit IFN-I responses. In this study, we report that SARS-CoV-2 NSP14 is a virus-encoded translation inhibitory factor which shuts down host protein synthesis, including synthesis of antiviral proteins. Our finding reveals a mechanism by which SARS-CoV-2 evades host antiviral responses. A comprehensive understanding of the strategies employed by SARS-CoV-2 to subvert host immune responses is critical for the design of next-generation antivirals and to prepare for future emerging viral pathogens. Abstract The ongoing COVID-19 pandemic has caused an unprecedented global health crisis. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19. Subversion of host protein synthesis is a common strategy that pathogenic viruses use to replicate and propagate in their host. In this study, we show that SARS-CoV-2 is able to shut down host protein synthesis and that SARS-CoV-2 nonstructural protein NSP14 exerts this activity. We show that the translation inhibition activity of NSP14 is conserved in human coronaviruses. NSP14 is required for virus replication through contribution of its exoribonuclease (ExoN) and N7-methyltransferase (N7-MTase) activities. Mutations in the ExoN or N7-MTase active sites of SARS-CoV-2 NSP14 abolish its translation inhibition activity. In addition, we show that the formation of NSP14−NSP10 complex enhances translation inhibition executed by NSP14. Consequently, the translational shutdown by NSP14 abolishes the type I interferon (IFN-I)-dependent induction of interferon-stimulated genes (ISGs). Together, we find that SARS-CoV-2 shuts down host innate immune responses via a translation inhibitor, providing insights into the pathogenesis of SARS-CoV-2. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the ongoing COVID-19 pandemic that has caused more than 120 million confirmed cases, resulting in more than 2.7 million deaths globally (https://covid19.who.int). SARS-CoV-2 belongs to the Coronaviridae family, in the genus Betacoronavirus, which also includes two highly pathogenic human coronaviruses, severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) (1). These human coronaviruses are associated with severe lower respiratory tract infection leading to severe and fatal respiratory syndromes in humans. Currently, there is an urgent need to better understand the molecular mechanisms of SARS-CoV-2 pathogenesis, which will help us to design better antivirals and next-generation vaccines. Replication of coronaviruses shuts down host protein synthesis in infected cells (2, 3). Multiple coronavirus proteins have been shown to hijack the host translation machinery to facilitate viral protein production. Despite the lack of sequence homology, coronavirus NSP1 proteins employ divergent mechanisms to suppress host protein expression (4). SARS-CoV NSP1 binds the small ribosomal subunit at the messenger RNA (mRNA) entry tunnel and inhibits mRNA translation (5, 6). Similarly, ribosome interaction and translation inhibition activity have been reported recently for SARS-CoV-2 NSP1 (7⇓⇓–10). Moreover, SARS-CoV accessory protein ORF7a and structural proteins spike (S) and nucleocapsid (N) proteins have been shown to inhibit protein synthesis (11⇓–13). The precise mechanisms of translation inhibition by these SARS-CoV proteins remain to be determined. Given the protein homology between SARS-CoV and SARS-CoV-2, it is likely that multiple SARS-CoV-2 viral proteins harbor translational regulation activity. SARS-CoV-2 has two large open reading frames (ORFs), ORF1a and ORF1b, encoding multiple nonstructural proteins (NSPs) involving every aspect of viral replication. ORF1a and ORF1b undergo proteolytic cleavage by viral-encoded proteinases to generate 16 mature NSPs, NSP1 to NSP16. Coronavirus NSP14 proteins are known to have 3′ to 5′ exoribonuclease (ExoN) activity and guanine-N7-methyltransferase activity (N7-MTase). The N-terminal ExoN domain is predicted to provide proofreading activity allowing removal of mismatched nucleotides introduced by the viral RNA-dependent RNA polymerase (14, 15). Given the large viral genome of coronaviruses, the proofreading activity of the ExoN domain is critical to maintain a high level of replication fidelity (16, 17). Recently, it has been shown that mutations in the active site and ZF motifs of the ExoN domain result in a lethal phenotype in SARS-CoV-2 and MERS-CoV (18). The C-terminal domain of NSP14 contains an S-adenosyl methionine (SAM)-dependent N7-MTase, which plays a critical role in viral RNA 5′ capping (19, 20). The 5′ cap facilitates viral mRNA stability and translation and prevents detection by host innate antiviral responses. SARS-CoV NSP14 forms a protein complex with NSP10, which is a zinc-binding protein with no reported enzymatic activity (20). NSP10 interacts with the N-terminal ExoN domain of NSP14 and enhances the ExoN activity but not the N7-MTase activity (14, 20). Notably, mutations in NSP10 that abolish the NSP14−NSP10 interaction result in a lethal phenotype in SARS-CoV (21). In this study, we investigated the ability of SARS-CoV-2 NSP14 to suppress host protein synthesis and the type I interferon (IFN-I) response. Similar to SARS-CoV infection (2), we found that SARS-CoV-2 shuts down host protein synthesis. As shown for SARS-CoV (5, 6), and, more recently, for SARS CoV-2 (7⇓–9), we confirmed that overexpression of NSP1 reduces protein synthesis in cells. In addition, we found that overexpression of NSP14 induces a near-complete shutdown in cellular protein synthesis. We also determined that the translation inhibition activity of NSP14 is conserved in several human coronaviruses. We demonstrated that mutations that inactivate either ExoN or N7-MTase enzymatic activities reverse translation inhibition mediated by NSP14. We also found that the formation of an NSP14−NSP10 protein complex enhances translation inhibition executed by NSP14 and showed that mutation of residues critical for this interaction abolishes this enhanced activity. Translation inhibition by NSP14 blocks IFN-I−dependent ISG induction, inhibiting the production of antiviral proteins. Our results provide mechanistic insights into the evasion of the innate immune responses by NSP14, a SARS-CoV-2 encoded translation inhibitor.
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Post by Admin on Nov 8, 2021 22:26:51 GMT
SARS-CoV-2 Infection Induces Translational Shutdown. Coronaviruses shut down host translation during viral replication (2, 3), and the newly emerged human coronavirus SARS-CoV-2 NSP1 protein was shown to inhibit translation (7⇓⇓–10). To further evaluate the effect of SARS-CoV-2 on host translation, we examined total protein synthesis during SARS-CoV-2 infection. Vero E6 cells were infected with SARS-CoV-2. At 24 h postinfection, total protein synthesis was measured by a puromycin incorporation assay. We observed that SARS-CoV-2 infection reduced protein synthesis (Fig. 1A). Notably, upon SARS-CoV-2 infection, two protein bands at ∼64 kDa, absent in uninfected cells and hence presumably viral proteins, were dominantly labeled by puromycin (Fig. 1A). These results suggest that SARS-CoV-2 infection shuts down cellular translation while promoting viral protein translation. We next examined whether the translational inhibition is restricted to the infected cells in the culture. Vero E6 cells were infected with recombinant SARS-CoV-2 expressing fluorescent protein mNeonGreen (CoV-2-mNG) (22). At 24 h postinfection, cellular protein synthesis was assessed by an O-propargyl puromycin (OP-Puro) labeling assay and confocal microscopy. We observed OP-Puro labeling in the uninfected cells, but not in the CoV-2-mNG−positive cells, suggesting that the protein synthesis inhibition is restricted to the SARS-CoV-2−infected cells (Fig. 1B). Fig. 1. SARS-CoV-2 infection inhibits cellular translation. (A) Vero E6 cells were infected with SARS-CoV-2 at the indicated MOIs. After 24 h of infection, cells were pulse labeled with puromycin for 15 min. Puromycin incorporation was determined by immunoblotting using anti-puromycin antibody (Puro). Expression of SARS-CoV-2 viral protein was determined using anti-SARS-CoV ORF3a antibody (ORF3a). (B) Confocal images of Vero E6 cells infected with recombinant SARS-CoV-2 expressing mNeonGreen (CoV-2-mNG) (22) at an MOI of 0.5. After 24 h of infection, cells were pulse labeled with OP-Puro for 1 h, fixed, fluorescently labeled by the Click chemistry reaction, and stained by Hoechst. (Scale bars, 10 µm.) SARS-CoV-2 NSP14 Inhibits Cellular Translation. It has been reported that SARS-CoV-2 NSP1 inhibits translation. However, overexpression of NSP1 in cells showed only moderate translation inhibition (∼50%) (7, 8). Given the difference in the efficiency of translation inhibition between SARS-CoV-2 infection (Fig. 1) and NSP1 overexpression, we considered the possibility that other viral proteins contributed to translational regulation. To investigate this, we examined translation in 293T cells overexpressing SARS-CoV-2 proteins, using OP-Puro labeling (Fig. 2A). The results showed that NSP1, NSP5, NSP14, and NSP15 significantly reduced OP-Puro labeling, suggesting that multiple SARS-CoV-2 proteins are involved in translation inhibition. Consistent with the previous studies (7, 8), we showed that NSP1 inhibited translation in 293T cells by ∼50% (Fig. 2A). However, overexpression of NSP14 reduced OP-Puro labeling even more significantly, by ∼75% (Fig. 2A), suggesting that NSP14 is a potent translation inhibitor. Inhibition was also apparent by a puromycin incorporation assay and by confocal microscopy of the OP-Puro−labeled 293T cells (Fig. 2 B and C). We further confirmed the translation inhibition activity of NSP14 by polysome profiling using sucrose density gradient centrifugation. Compared with empty vector control, the polysome profiles showed a drastic decrease in translating polyribosomes and an increase in 80S ribosomes in the presence of NSP14 (Fig. 2D, red line), indicating global inhibition of translation. Consistent with a previous study, NSP1 induced a similar shift (8), although this was less pronounced than that observed with NSP14 (Fig. 2D, blue line). These decreases in translation efficiency were quantified by the ratio of actively translating polysomes versus monosomes (P/M ratio), showing that NSP14 significantly inhibits cellular translation (Fig. 2E). Fig. 2. SARS-CoV-2 NSP14 inhibits cellular translation. (A) The 293T cells were transfected with plasmids encoding SARS-CoV-2 viral proteins. After 24 h of transfection, cells were pulse labeled with OP-Puro for 1 h, fixed, fluorescently labeled by the Click chemistry reaction, and analyzed by fluorescence-activated cell sorter (FACS). EV, empty vector. (B) The 293T cells were transfected with plasmids encoding indicated viral proteins for 24 h and puromycin labeled for 15 min. Puromycin incorporation was determined by immunoblotting using anti-puromycin antibody. (C) The 293T cells transfected and OP-Puro labeled as shown in A. Cells were stained by Hoechst and analyzed by confocal microscopy. (Scale bars, 50 µm.) (D) The 293T cells were transfected with plasmids encoding indicated viral proteins for 24 h. Cell lysates were cleared by centrifugation, loaded onto a 15 to 50% sucrose gradient, and subjected to ultracentrifugation. Absorbance was monitored at 254 nm to record the polysome profile. The monosome and polysome pools are indicated. (E) Quantification of polysome profile assay. Data are represented as the polysome-to-monosome (P/M) ratio. For A, B, and E, data are shown as mean ± SD of three biological repeats. *P < 0.05, **P < 0.01, ***P < 0.001 by unpaired Student’s t test. SARS-CoV-2 NSP1 inhibits nuclear export of host mRNA by poly(A) RNA nuclear retention and degradation in the cytoplasm (23). To determine whether NSP14 regulates mRNA stability and nuclear export, we performed RNA fluorescence in situ hybridization (FISH) to detect poly(A) RNA in 293T cells (SI Appendix, Fig. S1). The results showed that NSP14 has no effect on the poly(A) RNA distribution between the nucleus and cytoplasm (SI Appendix, Fig. S1A), nor on the total intracellular poly(A) RNA level (SI Appendix, Fig. S1B). To further examine whether NSP14 inhibits translation by inhibiting transcription, we coexpressed NSP14 with an EGFP reporter plasmid. Consistent with the results that measured global translation, we observed a significant reduction in EGFP fluorescence, quantified by flow cytometry, in the presence of NSP14, but not in the presence of NSP10 (SI Appendix, Fig. S2A). No significant change in the EGFP mRNA level was detected by qRT-PCR in the presence of either NSP14 or NSP10 (SI Appendix, Fig. S2B). Together, these results provide evidence that the translation inhibition by NSP14 is not a result of mRNA degradation or unpaired nuclear export. transfection of the human coronavirus NSP14 constructs resulted in a dose-dependent inhibition of puromycin incorporation (SI Appendix, Fig. S3). These results suggest that the NSP14 proteins of human coronaviruses share a critical role in translation inhibition during coronavirus infection.
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Post by Admin on Nov 9, 2021 4:47:59 GMT
Human Coronavirus NSP14 Proteins Inhibit Cellular Translation. NSP14 is a highly conserved viral protein in coronaviruses. In human betacoronaviruses, NSP14 of SARS-CoV-2 exhibits 99% and 77% amino acid sequence similarity to that of SARS-CoV and MERS-CoV, respectively (Fig. 3A). NSP14 of SARS-CoV-2 also shows ∼70% amino acid sequence similarity to NSP14 of HCoV-229E (human alphacoronavirus) and infectious bronchitis virus (IBV; an avian gammacoronavirus) (Fig. 3A). Given this homology, we asked whether NSP14 proteins of different coronaviruses could mediate translation inhibition. We determined cellular translation activity using OP-Puro labeling of 293T cells overexpressing HA-tagged NSP14 proteins from different coronaviruses. The results showed that all three human betacoronavirus NSP14 proteins inhibited translation (Fig. 3B). Moreover, alphacoronavirus HCoV-229E NSP14 exhibited a similar degree of translation inhibition, whereas IBV NSP14 only slightly reduced translation (Fig. 3B). This result was replicated using a puromycin incorporation assay (Fig. 3 C and D). Notably, transfection of equal amounts of plasmid DNA resulted in a much lower IBV NSP14 protein level than that of human coronaviruses (Fig. 3C). Therefore, 293T cells were transfected with increasing amounts of plasmid DNA and subjected to a puromycin incorporation assay. We found that IBV NSP14 was poorly expressed even with high amounts of plasmid DNA transfection, which likely resulted in weak translation inhibition (SI Appendix, Fig. S3). In contrast, transfection of the human coronavirus NSP14 constructs resulted in a dose-dependent inhibition of puromycin incorporation (SI Appendix, Fig. S3). These results suggest that the NSP14 proteins of human coronaviruses share a critical role in translation inhibition during coronavirus infection. Fig. 3. Human coronavirus NSP14 proteins inhibit cellular translation. (A) Comparison of the amino acid sequences of coronavirus NSP14. Sequence similarity of NSP14 between avian gammacoronavirus (IBV), human alphacoronavirus (HCoV-229E; 229E), and human betacoronaviruses (MERS-CoV; MERS, SARS-CoV; CoV-1, SARS-CoV-2; CoV-2). (B) The 293T cells were transfected with plasmids encoding different HA-tagged NSP14. After 24 h of transfection, cells were pulse labeled with OP-Puro for 1 h, fixed, fluorescently labeled by the Click chemistry reaction, and analyzed by FACS. (C) The 293T cells were transfected for 24 h and puromycin labeled for 15 min. Puromycin incorporation was determined by immunoblotting using anti-puromycin antibody (Puro). HA-tagged NSP14 proteins were detected by anti-HA antibody (HA). (D) Quantification of puromycin incorporation assay shown in C. For B and D, data are shown as mean ± SD of three biological repeats. *P < 0.05, **P < 0.01, ***P < 0.001 by unpaired Student’s t test; ns, not significant. Mutations Reducing ExoN and N7-MTase Activities Reverse Translation Inhibition. NSP14 is a member of the superfamily of DEDDh exoribonucleases, which contain five conserved active-site residues critical for ExoN activity (15, 20, 24). Additionally, there are two zinc finger (ZF) motifs, which contribute to the structural stability and catalytic activity of the ExoN domain (20). To determine whether the ExoN activity of SARS-CoV-2 NSP14 is responsible for translation inhibition, we generated a catalytically inactive NSP14 mutant, H268A (M2) (Fig. 4A). Using OP-Puro labeling and puromycin incorporation assays, we showed that H268A mutation (M2) in the ExoN active site abolished the translation inhibition activity (Fig. 4 B–D). These results were confirmed by polysome profiling in 293T cells (Fig. 4E). We then generated a ZF 2 motif mutant, C261A (M3) that structurally destabilizes the ExoN activity (20). We found that, although the C261A mutation is distal from the active site, M3 failed to inhibit translation (Fig. 4 B–D). In contrast, the mutant M1, with an alanine substitution at the noncatalytic D243 residue, D243A, retained the translation inhibition activity (Fig. 4 B–D). These results suggest that a functional ExoN domain is required to inhibit translation. Finally, we asked whether the N7-MTase activity is required. We found that D331A/G333A double mutation (M4) in the N7-MTase active site (20) abolished the translational inhibition activity of NSP14 (Fig. 4 B–D), suggesting that the N7-MTase activity is also required for translation inhibition. Notably, transfection of equal amounts of plasmids encoding NSP14 and its mutants resulted in much lower NSP14 and M1 protein levels than the other mutants (Fig. 4B). However, no such reduction was found on the NSP14 mRNA level (SI Appendix, Fig. S2C). This indicates that NSP14 inhibits not only cellular protein synthesis but its own mRNA translation, which has been shown in overexpression of SARS-CoV and SARS-CoV-2 NSP1 (6⇓–8). Together, these data are consistent with the suggestion that both the ExoN and N7-MTase activities of SARS-CoV-2 NSP14 are required to inhibit translation. Fig. 4. ExoN and N7-MTase are required for translation inhibition activity of NSP14. (A) Crystal structure of SARS-CoV NSP10−NSP14 complex (Protein Data Bank ID code 5NFY). NSP10 is shown in gray. N-terminal ExoN and C-terminal N7-MTase domains are in blue and green, respectively. NSP14 mutants are described below, in the table. The mutation residues are highlighted in the structure. (B) The 293T cells were transfected with plasmids encoding WT or NSP14 mutants for 24 h and puromycin labeled for 15 min. Puromycin incorporation was determined by immunoblotting using anti-puromycin antibody (Puro). HA-tagged NSP14 proteins were detected by anti-HA antibody (HA). (C) Quantification of puromycin incorporation assay shown in B. (D) The 293T cells were transfected with plasmids encoding WT or NSP14 mutants. After 24 h of transfection, cells were pulse labeled with OP-Puro for 1 h, fixed, fluorescently labeled, and analyzed by FACS. (E) The 293T cells were transfected with plasmids encoding NSP14 or M2 mutant for 24 h. Cell lysates were cleared by centrifugation, loaded onto a 15 to 50% sucrose gradient, and subjected to ultracentrifugation. Absorbance was monitored at 254 nm to record the polysome profile. The monosome and polysome pools are indicated. For C and D, data are shown as mean ± SD of three biological repeats. *P < 0.05, **P < 0.01, ***P < 0.001 by unpaired Student’s t test.
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