NSP14 Protein Weakens SARS-CoV-2

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NSP14 Protein Weakens SARS-CoV-2 Nov 9, 2021 21:16:01 GMT

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Post by Admin on Nov 9, 2021 21:16:01 GMT

NSP10 Enhances Translation Inhibition Activity of NSP14.
SARS-CoV NSP14 forms a protein complex with NSP10 (Fig. 4A) (20). NSP10 is a central modulator in the replication and transcription complex of coronaviruses (21). Moreover, NSP10 activates the enzymatic activity of NSP14 and NSP16 through protein−protein interaction (20, 21). NSP10 interacts with the N-terminal ExoN domain of NSP14, selectively enhancing the ExoN activity but not the N7-MTase activity (14, 25). Given the high protein sequence homology between SARS-CoV and SARS-CoV-2 (SI Appendix, Figs. S4 and S5), we hypothesized that SARS-CoV-2 NSP10 and NSP14 might also interact with each other. Immunoprecipitation (IP) experiments involving 293T cells expressing both NSP10 and NSP14 revealed that the two proteins coprecipitated (Fig. 5A). We then examined whether NSP10 interaction affects the translation inhibition activity of NSP14. Using the puromycin incorporation assay, we showed that, although NSP14 inhibited translation as expected, coexpression of NSP10 further enhanced the inhibition (Fig. 5 B and C). Notably, NSP10 coexpression significantly increased the NSP14 protein level (Fig. 5B), consistent with the idea that the formation of the complex increases NSP14 protein stability and consequently enhances the observed inhibition of translation. Next, we investigated whether the complex formation can rescue the translation inhibition activity of NSP14 mutants. Using puromycin incorporation assays, we found that coexpression of NSP10 with wild-type (WT) NSP14 and the mutants increased the translation inhibition activity of WT NSP14 and the active mutant M1 (Fig. 5D and SI Appendix, Fig. S6A). Notably, we found that the translation inhibition activity of M3, known to have a structurally destabilized N-terminal ExoN domain in SARS-CoV NSP14, was restored by coexpression of NSP10 (Fig. 5D and SI Appendix, Fig. S6A). In contrast, coexpression with NSP10 failed to rescue the translation inhibition activities of M2 and M4 bearing mutations in the active sites (Fig. 5D and SI Appendix, Fig. S6A). These results suggest that coexpression with NSP10 enhances the translation inhibition activity of NSP14 primarily by association and structural stabilization.

Fig. 5.
NSP14−NSP10 complex formation enhances the translation inhibition activity of NSP14. (A) The 293T cells were transfected with plasmids encoding indicated proteins for 24 h. Cell lysates were subjected to IP using mouse anti-HA antibody and followed by immunoblotting with rabbit anti-HA and anti-FLAG antibodies. WCE, whole cell extract. (B) The 293T cells were transfected with plasmids encoding NSP10 or NSP14 for 24 h and puromycin labeled for 15 min. Puromycin incorporation was determined by immunoblotting using anti-puromycin antibody (Puro). HA-tagged NSP14 and FLAG-tagged NSP10 proteins were detected by anti-HA and anti-FLAG antibodies, respectively. (C) Quantification of puromycin incorporation assay shown in B. (D) The 293T cells were transfected for 24 h and puromycin labeled for 15 min. Puromycin incorporation was determined by immunoblotting. (E) The 293T cells were cotransfected with HA-tagged NSP14 and FLAG-tagged NSP10 or its mutants for 24 h. Cell lysates were subjected to IP using mouse anti-HA antibody and followed by immunoblotting with rabbit anti-HA and anti-FLAG antibodies. (F) The 293T cells were transfected for 24 h and puromycin labeled for 15 min. Puromycin incorporation was determined by immunoblotting. For C, D, and F, 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.

Several residues in the SARS-CoV NSP14−NSP10 interface have been reported to be required for the protein−protein interaction and activation of the ExoN activity of NSP14, including the amino acid residues Lys43 and His80 of NSP10 (21). We therefore generated two SARS-CoV-2 NSP10 mutants bearing alanine substitutions at these residues (K43A and H80A) (SI Appendix, Fig. S6 B and C). Consistent with the SARS-CoV study (21), IP assays showed that NSP10 (K43A) partially reduced the NSP10−NSP14 interaction, whereas NSP10 (H80A) completely lost the NSP14−NSP10 interaction (Fig. 5E). Consequently, as shown by the puromycin incorporation assay, coexpression of WT NSP14 with NSP10 (H80A) mutant did not enhance the translation inhibition, whereas NSP10 (K43A) mutant induced a moderate increase in translation inhibition (Fig. 5F and SI Appendix, Fig. S6D). Moreover, we found that the NSP10 mutants failed to enhance the translation inhibition activity of the structurally destabilized NSP14 mutant M3 (Fig. 5F and SI Appendix, Fig. S6D). Together, the results suggest that NSP10−NSP14 interaction enhances the translation inhibition activity.

NSP14 Inhibits IFN-Dependent ISG Induction.
SARS-CoV-2 NSP14 overexpression inhibits the production of IFN-beta and IFN-stimulated genes (ISGs) (26⇓–28). Consistent with these studies, we showed, by immunoblot analysis, that SARS-CoV-2 NSP14 overexpression suppresses endogenous ISG stimulation in response to IFN-I in Vero E6 cells (Fig. 6A). In contrast, NSP14 did not affect the induction of ISGs (Fig. 6B). As reported in the literature (8), NSP1 also inhibited ISG protein synthesis but not the mRNA (Fig. 6 A and B). This phenotype was examined for additional ISGs in 293T cells (Fig. 6C). Consistently, the results showed that NSP14 strongly inhibits the protein synthesis of antiviral ISGs (viperin, TRIM21, ISG15) and ISGs functioning in RNA sensing and signaling (retinoic acid-inducible gene I [RIG-I], MDA5, STING) in the IFN-I response (Fig. 6C). A recent preprint reported that, although SARS-CoV-2 infection up-regulates many ISGs on the mRNA level, their translation is impaired (29). Therefore, to determine whether the translation inhibition activity of NSP14 is responsible for inhibiting ISG expression, we evaluated the effect of the NSP14 mutants on the expression of endogenous ISGs in 293T cells. We found that M2 and M4 failed to inhibit the expression of ISGs, suggesting that inhibition of endogenous ISG expression by NSP14 is a result of its translation inhibition activity (Fig. 6D).

Fig. 6.
NSP14 inhibits the protein expression of ISGs. (A) Vero E6 cells were transfected with plasmids encoding NSP1 or NSP14 for 24 h and treated with IFN-I for 18 h. The expression of IFN-stimulated proteins was determined by immunoblotting with antibodies against depicted proteins. (B) The induction of ISGs on mRNA level from A were determined by qRT-PCR with primers against genes encoding RIG-I and viperin proteins (RSAD2). (C) The 293T cells were transfected with HA-tagged NSP14 for 24 h and treated with IFN-I for 18 h. The induction of ISGs was determined by immunoblotting with antibodies against depicted proteins. Asterisk (*) indicates nonspecific protein bands. (D) The 293T cells were transfected with plasmids encoding NSP1 or NSP14 for 24 h and treated with IFN-I for 18 h. The expression of IFN-stimulated proteins of ISGs was determined by immunoblotting with antibodies against depicted proteins. For B, data are shown as mean ± SD of six biological repeats by unpaired Student’s t test.

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NSP14 Protein Weakens SARS-CoV-2 Nov 9, 2021 21:38:40 GMT

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Post by Admin on Nov 9, 2021 21:38:40 GMT

Discussion
Similar to other coronaviruses (4), SARS-CoV-2 NSP1 was found to inhibit translation in in vitro translation systems by blocking the mRNA entry tunnel of the small ribosomal subunit, but it only exhibits moderate translation inhibition activity in cells (7, 8). K164A/H165A double mutations abolish the host gene suppression activities of NSP1 (7, 8). However, recombinant SARS-CoV bearing the mutations in NSP1 only partially inhibits the translation inhibition effect during viral replication (2). Moreover, the SARS-CoV mutant shows no difference in viral replication compared to WT virus (2). These observations suggest that multiple SARS-CoV-2 proteins may contribute to a translation shutdown. Here, using nascent peptide labeling assays, we showed that SARS-CoV-2 infection dramatically shuts down host translation (Fig. 1). Screening identified SARS-CoV-2 NSP14 as a novel inhibitor of host translation (Fig. 2). Using polysome profiling and nascent protein labeling coupled with flow cytometry and confocal microscopy, we found that NSP14 induces a more drastic reduction in host protein synthesis than NSP1 (Fig. 2). Polysome profiling in the presence of NSP14 exhibited a typical translation inhibition profile with an increase in 80S ribosome population and decrease in polysomes (Fig. 2 D and E). We also examined whether NSP14 regulates mRNA stability or nuclear export, using RNA FISH to detect poly(A) RNA. In cells expressing NSP14, unlike those expressing NSP1, we observed no effect on total intracellular poly(A) RNA levels, or on the poly(A) RNA distribution between the nucleus and cytoplasm, supporting a direct role for NSP14 in translation inhibition (SI Appendix, Fig. S1). Future experiments to determine the effect of NSP14-mediated translation inhibition on viral replication will require the generation of an appropriate recombinant virus.

Coronaviruses consist of four genera: alpha, beta, gamma, and delta. Human coronaviruses HCoV-229E and HCoV-NL63 belong to the alphacoronavirus genus, and HCoV-HKU1, HCoV-OC43, SARS-CoV, MERS-CoV, and SARS-CoV-2 belong to the betacoronavirus genus (1). Coronavirus NSP14 is a bifunctional enzyme, with both ExoN and N7-MTase activities requiring highly conserved residues in each active site. We showed that NSP14 proteins of four human coronaviruses, including three highly pathogenic betacoronaviruses, are able to inhibit host protein synthesis, which suggests that this role of NSP14 is conserved among these coronaviruses. While NSP14 proteins of HCoV-229E, SARS-CoV, and SARS-CoV-2 dramatically inhibit translation, MERS-CoV NSP14 translation inhibition was less pronounced (Fig. 3 C and D). In contrast, despite the high protein homology with human coronaviruses (Fig. 3A), NSP14 of the avian gammacoronavirus IBV only slightly inhibits translation (Fig. 3 C and D). Given that IBV is a host-specific coronavirus (30), we speculate that this relatively weak inhibition may result from low IBV NSP14 expression in human cells (SI Appendix, Fig. S13) and perhaps the lack of host-specific cofactors (30). Together, these observations probably reflect a critical role of translation inhibition during the replication of human coronaviruses.

Identification of NSP14 as a translation inhibitory protein was unexpected given that it has been characterized as a bifunctional enzyme. The N-terminal domain is a member of the superfamily of DEDDh ExoNs, which contains five conserved catalytic residues required for ExoN activity (15, 20, 24). These residues are essential for viral replication, as recombinant coronaviruses bearing ExoN active-site mutations are nonviable (18, 24). In this study, we investigated the role of the ExoN activity in translation inhibition by alanine substitution at residues D243 and H268 in the ExoN domain. Both residues were first reported as catalytic residues (24), whereas the SARS-CoV NSP14 crystal structure revealed that, despite being in close proximity, D243 is not a catalytic residue (20). While mutations at D243 and H268 abolish the ExoN activity of recombinant SARS-CoV and MERS-CoV NSP14 proteins (18⇓⇓–21), we found that the catalytic H268A mutation (M2) in the active site abolishes the translation inhibition activity, but D243A mutation (M1) has no effect (Fig. 4). Nevertheless, the H268A mutation result suggests a critical role for the ExoN domain in translation inhibition.

The ExoN domain has two ZF motifs, which contribute to the structural stability and catalytic activity of ExoN domain (20). Recombinant SARS-CoV NSP14 proteins bearing mutations in the ZF motifs abolish the ExoN activity, and similar mutations impair MERS-CoV replication (18, 20). Here, we found that C261A (M3), mutated in the ZF2 motif, does not inhibit translation (Fig. 4). Notably, the catalytic ExoN mutation H268A (M2) is in the ZF2 motif. These results suggest an important role of the ZF2 motif in translation inhibition activity of NSP14. Intriguingly, coexpression with NSP10 rescues the translation inhibition activity of M3 mutant (Fig. 5D). However, it has been shown that C261A mutation abolishes the ExoN activity even in the presence of NSP10 (20). This suggests that the structural integrity of the ExoN domain is essential for translation inhibition in a way that discriminates this activity from the ExoN activity.

Mutations in the ExoN and N7-MTase domains attenuate the in vitro RNA degradation and methyltransferase activity, respectively (14, 19, 31). Moreover, mutations in the ExoN domain do not interfere with the N7-MTase activity, and vice versa (18, 19, 31). The C-terminal N7-MTase domain is composed of a canonical SAM binding motif I that is responsible for viral RNA 5′ cap formation (19, 20). In vitro biochemical assays showed that mutations in the SAM binding motif of SARS-CoV and MERS-CoV NSP14 proteins result in inactivation of the N7-MTase activity (18⇓–20, 31). Moreover, N7-MTase mutation impairs the replication of betacoronavirus murine hepatitis virus and results in delayed kinetics of viral mRNA translation in vitro (32). Here, we found that the N7-MTase mutant (M4) bearing a D331A/G333A double mutation in the SAM binding motif fails to inhibit translation (Fig. 4). Thus we have identified mutations in both ExoN and N7-MTase domains of SARS-CoV-2 NSP14 that abolish the translation inhibition activity, suggesting that both domains may function cooperatively to execute translation inhibition. It remains to be determined exactly how these domains combine to contribute to translation inhibition.

NSP10 is a central modulator in the coronavirus replication and transcription complex but has no reported specific enzymatic activity itself. It activates the enzymatic activity of both NSP14 and NSP16 by protein−protein interaction (20, 21). NSP10 interacts with the N-terminal ExoN domain of SARS-CoV NSP14 and strongly enhances the ExoN activity but not the N7-MTase activity in vitro (14, 25). Notably, mutations in NSP10 that abolish the NSP10−NSP14 interaction inactivate SARS-CoV (21). We show here that SARS-CoV-2 NSP10 also forms a protein complex with NSP14 (Fig. 5A). While this interaction is not required for NSP14-mediated translation inhibition, it substantially stabilizes NSP14 expression, and, as a consequence, translation inhibition is significantly enhanced (Fig. 5 and SI Appendix, Fig. S4).

The IFN-I response is the first line of defense against viral invasion, leading to the production of hundreds of ISGs, many of which play an important role in the antiviral responses.

Coronavirus infections are sensed by a cytosolic pattern recognition receptor, RIG-I, which activates this defense system (33, 34). Replication of human betacoronaviruses, including both SARS-CoV and SARS-CoV-2, is sensitive to IFN-I pretreatment (35, 36), and multiple betacoronavirus proteins have been shown to antagonize IFN-I production and downstream antiviral ISG expression (37⇓–39). Recently, three independent studies using luciferase reporter assays identified multiple SARS-CoV-2 proteins as IFN-I antagonists, including NSP14 (26⇓–28). A recent unreviewed preprint reported that, although SARS-CoV-2 infection dramatically up-regulates numerous ISGs, their translation is impaired (29). Consistently, our results indicate that NSP14 shuts down the expression of endogenous antiviral ISGs via its translation inhibition activity (Fig. 6). Both the ExoN and N7-MTase activities of NSP14 play critical roles in counteracting the IFN-I responses during the replication of coronaviruses (32, 40), and, consistent with this, we found that mutations in the ExoN and N7-MTase domains abolish inhibition of ISG induction (Fig. 6D). Although the translation of ISGs can be selectively attenuated by dengue virus-2 infection (41), SARS-CoV-2 NSP1 inhibits the synthesis of IFN-stimulated proteins by global translation inhibition (8). In addition to NSP1, we found that SARS-CoV-2 NSP14 inhibits the protein expression of a variety of ISGs via its global translation inhibition activity (Fig. 6C), which provides an additional layer of protection against the IFN response. This redundancy of ISG antagonism has been reported for coronaviruses (28, 42).

It remains to be determined how SARS-CoV-2 overcomes the translation shutdown induced by the NSPs for the production of its own viral proteins. We speculate that, as shown for SARS-CoV, a common sequence/structure in the 5′ untranslated region of viral mRNAs facilitates efficient translation of viral mRNAs (43). Nevertheless, this work provides evidence that the likely mechanism underlying the involvement of NSP14 in immune evasion lies in its capacity to interfere with host protein generation. Thus, NSP14 both ensures the fidelity of viral mRNA translation and blocks the translation of host mRNAs. Furthermore, the mutagenesis study of NSP14 enzymatic activity and NSP10–NSP14 interaction demonstrates several unique niches that may provide a starting point for development of potent antiviral drugs.