Conditional ablation of CD205+ conventional dendritic cells impacts the regulation of T-cell immunity and homeostasis in vivo

Dendritic cells (DCs) are composed of multiple subsets that play a dual role in inducing immunity and tolerance. However, it is unclear how CD205(+) conventional DCs (cDCs) control immune responses in vivo. Here we generated knock-in mice with the selective conditional ablation of CD205(+) cDCs. CD205(+) cDCs contributed to antigen-specific priming of CD4(+) T cells under steady-state conditions, whereas they were dispensable for antigen-specific CD4(+) T-cell responses under inflammatory conditions. In contrast, CD205(+) cDCs were required for antigen-specific priming of CD8(+) T cells to generate cytotoxic T lymphocytes (CTLs) mediated through cross-presentation. Although CD205(+) cDCs were involved in the thymic generation of CD4(+) regulatory T cells (Tregs), they maintained the homeostasis of CD4(+) Tregs and CD4(+) effector T cells in peripheral and mucosal tissues. On the other hand, CD205(+) cDCs were involved in the inflammation triggered by Toll-like receptor ligand as well as bacterial and viral infections. Upon microbial infections, CD205(+) cDCs contributed to the cross-priming of CD8(+) T cells for generating antimicrobial CTLs to efficiently eliminate pathogens, whereas they suppressed antimicrobial CD4(+) T-cell responses. Thus, these findings reveal a critical role for CD205(+) cDCs in the regulation of T-cell immunity and homeostasis in vivo.     

Differential function of the NACHT-LRR (NLR) members Nod1 and Nod2 in arthritis

The pathogenesis of chronic joint inflammation remains unclear, although the involvement of pathogen recognition receptors has been suggested recently. In the present article, we describe the role of two members of the NACHT-LRR (NLR) family, Nod1 (nucleotide-binding oligomerization domain) and Nod2 in a model of acute joint inflammation induced by intraarticular injection of Streptococcus pyogenes cell wall fragments. Here, we show that Nod2 deficiency resulted in reduced joint inflammation and protection against early cartilage damage. In contrast, Nod1 gene-deficient mice developed enhanced joint inflammation with concomitant elevated levels of proinflammatory cytokines and cartilage damage, consistent with a model in which Nod1 controls the inflammatory reaction. To explore whether the different function of Nod1 and Nod2 occurs also in humans, we exposed peripheral blood mononuclear cells (PBMCs) carrying either Nod1ins/del or Nod2fs mutation with SCW fragments in vitro. Production of both TNFalpha and IL-1beta was clearly impaired in PBMCs carrying the Nod2fs compared with PBMCs isolated from healthy controls. In line with results in Nod1 gene-deficient mice, PBMCs from individuals bearing a newly described Nod1 mutation produced enhanced levels of proinflammatory cytokines after 24-h stimulation with SCW fragments. These data indicate that the NLR family members Nod1 and Nod2 have different functions in controlling inflammation, and that intracellular Nod1-Nod2 interactions may determine the severity of arthritis in this experimental model. Whether a distorted balance between the function of Nod1 and/or Nod2 is involved in the pathogenesis of human autoinflammatory or autoimmune disease, such as rheumatoid arthritis, remains to be elucidated.     

Expression of the Sendai (murine parainfluenza) virus C protein alleviates restriction of measles virus growth in mouse cells

Measles virus (MV), a human pathogen, uses the signaling lymphocyte activation molecule (SLAM) or CD46 as an entry receptor. Although several transgenic mice expressing these receptors have been generated as small animal models for measles, these mice usually have to be made defective in IFN-α/β signaling to facilitate MV replication. Similarly, when functional receptors are expressed by transfection, mouse cells do not allow MV growth as efficiently as primate cells. In this study, we demonstrate that MV efficiently grows in SLAM-expressing mouse cells in which the Sendai virus (SeV) C protein is transiently expressed. We developed a SLAM-expressing mouse cell line whose genome also encodes the SeV C protein downstream of the sequence flanked with loxP sequences. When this cell line was infected with the recombinant MV expressing the Cre recombinase, the SeV C protein was readily expressed. Importantly, the Cre recombinase-encoding MV grew in this cell line much more efficiently than it did in the parental cell. The minigenome assay demonstrated that the SeV C protein does not modulate MV RNA synthesis. Analyses using the mutant proteins with the defined functional defects revealed that the IFN-antagonist function, but not the budding-accelerating function, of the SeV C protein was critical for supporting efficient MV growth in mouse cells. Our results indicate that insufficient IFN antagonism can be an important determinant of the host range of viruses, and the system described here may be useful to overcome the species barrier of other human viruses.     

Critical coordination of innate immune defense against Toxoplasma gondii by dendritic cells responding via their Toll-like receptors

Toll-like receptors (TLRs) play an important role in host defense against a variety of microbial pathogens. We addressed the mechanism by which TLRs contribute to host defense against the lethal parasite Toxoplasma gondii by using mice with targeted inactivation of the TLR adaptor protein myeloid differentiation primary response gene 88 (MyD88) in different innate cell types. Lack of MyD88 in dendritic cells (DCs), but not in macrophages or neutrophils, resulted in high susceptibility to the T. gondii infection. In the mice deficient in MyD88 in DCs, the early IL-12 response by DCs was ablated, the IFN-γ response by natural killer cells was delayed, and the recruited inflammatory monocytes were incapable of killing the T. gondii parasites. The T-cell response, although attenuated in these mice, was sufficient to eradicate the parasite during the chronic stage, provided that defects in DC activation were compensated by IL-12 treatment early after infection. These results demonstrate a central role of DCs in orchestrating the innate immune response to an intracellular pathogen and establish that defects in pathogen recognition by DCs can predetermine sensitivity to infection.     

Aspartate-glutamate-alanine-histidine box motif (DEAH)/RNA helicase A helicases sense microbial DNA in human plasmacytoid dendritic cells

Toll-like receptor 9 (TLR9) senses microbial DNA and triggers type I IFN responses in plasmacytoid dendritic cells (pDCs). Previous studies suggest the presence of myeloid differentiation primary response gene 88 (MyD88)-dependent DNA sensors other than TLR9 in pDCs. Using MS, we investigated C-phosphate-G (CpG)-binding proteins from human pDCs, pDC-cell lines, and interferon regulatory factor 7 (IRF7)-expressing B-cell lines. CpG-A selectively bound the aspartate-glutamate-any amino acid-aspartate/histidine (DExD/H)-box helicase 36 (DHX36), whereas CpG-B selectively bound DExD/H-box helicase 9 (DHX9). Although the aspartate-glutamate-alanine-histidine box motif (DEAH) domain of DHX36 was essential for CpG-A binding, the domain of unknown function 1605 (DUF1605 domain) of DHX9 was required for CpG-B binding. DHX36 is associated with IFN-α production and IRF7 nuclear translocation in response to CpG-A, but DHX9 is important for TNF-α and IL-6 production and NF-κB activation in response to CpG-B. Knocking down DHX9 or DHX36 significantly reduced the cytokine responses of pDCs to a DNA virus but had no effect on the cytokine responses to an RNA virus. We further showed that both DHX9 and DHX36 are localized within the cytosol and are directly bound to the Toll-interleukin receptor domain of MyD88 via their helicase-associated domain 2 and DUF domains. This study demonstrates that DHX9/DHX36 represent the MyD88-dependent DNA sensors in the cytosol of pDCs and suggests a much broader role for DHX helicases in viral sensing.     

肺脏基质细胞分泌的血管内皮生长因子对树突状细胞分化及功能的影响

目的 观察小鼠肺脏基质细胞分泌的血管内皮生长因子(VEGF)对成熟树突状细胞(mDC)分化和功能及免疫耐受的影响.方法 建立肺脏基质细胞/树突状细胞共培养体系作为对照组,体系中加入VEGF抗体作为实验组(VEGF抗体组).反转录聚合酶链反应法检测肺脏基质细胞微环境中细胞因子的表达;透射电镜观察细胞形态.流式细胞术检测细...     

Activation and function of hepatic NK cells in hepatitis B infection: an underinvestigated innate immune response

Natural killer (NK) cells are abundant in the normal liver, accounting for around one-third of intrahepatic lymphocytes and are important in the defence against hepatitis B virus (HBV) infection as innate immune responses. In this review, we discuss the mechanisms of hepatic NK cell activity against HBV. Whether directly activated by HBV infection or indirectly activated by other lymphocytes such as NKT cells or antigen-presenting cells (APCs), hepatic NK cells exert their anti-viral functions by natural cytotoxicity and production of high levels of cytokines. However, activated NK cells play an important role in regulating adaptive immune responses by interaction with other lymphocytes such as T, B and APCs. In addition, NK cells may contribute to the lymphocyte-mediated liver injury during HBV infection that was previously considered to be mediated only by CD8+ T cells or/and NKT cells.     

US3 protein kinase of HSV-1 cycles between the cytoplasm and nucleus and interacts with programmed cell death protein 4 (PDCD4) to block apoptosis

The U(S)3 protein kinase of herpes simplex virus 1 plays a key role in blocking apoptosis induced by viral gene products or exogenous agents. The U(S)3 protein kinase is similar to protein kinase A with respect to substrate range and specificity. We report that in the yeast two-hybrid system a domain of U(S)3 essential for antiapoptotic activity reacted with programmed cell death protein 4 (PDCD4). We report that U(S)3 interacts with PDCD4, that PDCD4 is posttranslationally modified in infected cells both in a U(S)3-dependent and -independent fashion, and that depletion of PDCD4 by siRNA blocked apoptosis induced by a Δα4 mutant virus. In infected cells, PDCD4 accumulates in the nucleus, whereas U(S)3 accumulates in the cytoplasm. Studies designed to elucidate the convergence of these proteins led to the discovery that U(S)3 protein kinase cycles between the nucleus and cytoplasm and that U(S)3 retains PDCD4 in infected cell nuclei.     

Signaling mechanisms in the restoration of impaired immune function due to diet-induced obesity

Our previous data have linked obesity with immune dysfunction. It is known that physical exercise with dietary control has beneficial effects on immune function and the comorbidities of obesity. However, the mechanisms underlying the improvement of immune function in obesity after physical exercise with dietary control remain unknown. Here we show that moderate daily exercise with dietary control restores the impaired cytokine responses in diet-induced obese (DIO) mice and improves the resolution of Porphyromonas gingivalis-induced periodontitis. This restoration of immune responses is related to the reduction of circulating free fatty acids (FFAs) and TNF. Both FFAs and TNF induce an Akt inhibitor, carboxyl-terminal modulator protein (CTMP). The expression of CTMP is also observed increased in bone marrow-derived macrophages (BMMΦ) from DIO mice and restored after moderate daily exercise with dietary control. Toll-like receptor 2 (TLR2), which increases CTMP induction by FFAs, is inhibited in BMMΦ from DIO mice or after either FFA or TNF treatment, but unexpectedly is not restored by moderate daily exercise with dietary control. Furthermore, BMMΦ from DIO mice display reduced histone H3 (Lys-9) acetylation and NF-κB recruitment to TNF, IL-10, and TLR2 promoters after P. gingivalis infection. However, moderate daily exercise with dietary control restores these defects at promoters for TNF and IL-10, but not for TLR2. Thus, metabolizing FFAs and TNF by moderate daily exercise with dietary control improves innate immune responses to infection in DIO mice via restoration of CTMP and chromatin modification.     

The innate immune response in calves to Boophilus microplus tick transmitted Babesia bovis involves type-1 cytokine induction and NK-like cells in the spleen

The innate immune response to Babesia bovis infection in cattle is age-related, spleen-dependent and, in stabilate inoculated calves, has type-1 characteristics, including the early induction of IL-12 and IFN-gamma. In this study with three calves, parameters of innate immunity were followed for 2 weeks after tick transmission of B. bovis. Each calf survived the acute disease episode without drug intervention, and responded with increased levels of plasma interferon-gamma and type-1 cytokine expression, monocyte/macrophage activation, and CD8+ cellular proliferation in the spleen. The proliferating CD8+ population consisted primarily of NK-like cells, and the expansion occurred in parallel with an increase in IL-15 mRNA expression in the spleen.     

Induction of innate immune response and absence of subsequent tolerance to dsRNA in biliary epithelial cells relate to the pathogenesis of biliary atresia

Background/Aims: Biliary epithelial cells (BECs) possess negative regulatory mechanisms of Toll‐like receptor (TLR)‐based tolerance to bacteria (e.g. endotoxin tolerance). Viral infections of the Reoviridae genus with a dsRNA genome are suspected to be part of the aetiology of biliary atresia (BA), but the negative biliary mechanisms remain unexplored. Methods: Cultured human intrahepatic BECs (HIBECs) pretreated with polyinosinic–polycytidylic acid [poly(I:C)] (a synthetic analogue of viral dsRNA) for 24 h were exposed to poly(I:C) in fresh medium. The activation of nuclear factor‐κB (NF‐κB) and the expression of myxovirus resistance protein A (MxA) and tumour necrosis factor‐related apoptosis‐inducing ligand (TRAIL) mRNAs were evaluated. Moreover, after the pretreatment, the transition of these molecules was examined in poly(I:C)‐free conditions. Results: Treatment with poly(I:C) significantly upregulated NF‐κB activity in fresh HIBECs, and pretreatment failed to show tolerance to poly(I:C). The production of MxA and TRAIL was also preserved. Moreover, upregulation in the pretreated HIBECs was well preserved in poly(I:C)‐free medium for at least 72 h. Conclusions: BECs fail to show tolerance to poly(I:C), and once innate immunity is activated it is sustained in poly(I:C)‐free conditions, suggesting that the initiation of the immune response to dsRNA in HIBECs and its presence after the clearance of virus are closely associated with the progression of BA.     

Kinase-independent function of checkpoint kinase 1 (Chk1) in the replication of damaged DNA

The checkpoint kinases Chk1 and ATR are broadly known for their role in the response to the accumulation of damaged DNA. Because Chk1 activation requires its phosphorylation by ATR, it is expected that ATR or Chk1 down-regulation should cause similar alterations in the signals triggered by DNA lesions. Intriguingly, we found that Chk1, but not ATR, promotes the progression of replication forks after UV irradiation. Strikingly, this role of Chk1 is independent of its kinase-domain and of its partnership with Claspin. Instead, we demonstrate that the ability of Chk1 to promote replication fork progression on damaged DNA templates relies on its recently identified proliferating cell nuclear antigen-interacting motif, which is required for its release from chromatin after DNA damage. Also supporting the importance of Chk1 release, a histone H2B-Chk1 chimera, which is permanently immobilized in chromatin, is unable to promote the replication of damaged DNA. Moreover, inefficient chromatin dissociation of Chk1 impairs the efficient recruitment of the specialized DNA polymerase η (pol η) to replication-associated foci after UV. Given the critical role of pol η during translesion DNA synthesis (TLS), these findings unveil an unforeseen facet of the regulation by Chk1 of DNA replication. This kinase-independent role of Chk1 is exclusively associated to the maintenance of active replication forks after UV irradiation in a manner in which Chk1 release prompts TLS to avoid replication stalling.     

Hypoxia-inducible factor 1-dependent expression of platelet-derived growth factor B promotes lymphatic metastasis of hypoxic breast cancer cells

Lymphatic dissemination from the primary tumor is a major mechanism by which breast cancer cells access the systemic circulation, resulting in distant metastasis and mortality. Numerous studies link activation of hypoxia-inducible factor 1 (HIF-1) with tumor angiogenesis, metastasis, and patient mortality. However, the role of HIF-1 in lymphatic dissemination is poorly understood. In this study, we show that HIF-1 promotes lymphatic metastasis of breast cancer by direct transactivation of the gene encoding platelet-derived growth factor B (PDGF-B), which has proliferative and chemotactic effects on lymphatic endothelial cells. Lymphangiogenesis and lymphatic metastasis in mice bearing human breast cancer orthografts were blocked by administration of the HIF-1 inhibitor digoxin or the tyrosine kinase inhibitor imatinib. Immunohistochemical analysis of human breast cancer biopsies demonstrated colocalization of HIF-1α and PDGF-B, which were correlated with lymphatic vessel area and histological grade. Taken together, these data provide experimental support for breast cancer clinical trials targeting HIF-1 and PDGF-B.     

Rapamycin combined with donor immature dendritic cells promotes liver allograft survival in association with CD4(+) CD25(+) Foxp3(+) regulatory T cell expansion

Aim: To determine whether donor immature dendritic cells (imDCs) combined with a short postoperative course of rapamycin (Rapa) has the ability to expand the CD4⁺CD25⁺Foxp3⁺ regulatory T (Treg) cells and prolong liver allograft survival. Methods: Orthotopic liver transplantation (OLT) was performed from Lewis rats to Brown Norway recipients. Three days before transplantation, animals were injected intravenously with 2 × 10⁶ donor bone marrow‐derived imDCs. Recipient rats (the combined treated group) also received Rapa for 7 d after liver transplantation. Additional groups received either imDCs alone, Rapa alone, or saline alone. Every six recipients from each group were killed at 14 days, 28 days after OLT. The changes of CD4⁺CD25⁺Foxp3⁺ Treg cells in peripheral blood and spleen, histological changes of liver grafts, and serum cytokine levels were investigated. The other six recipients were left in each group to observe the animal survival. Results: Donor imDCs followed by a short postoperative course of Rapa induced long‐term allograft survival. The percentage of CD4⁺CD25⁺Foxp3⁺ Treg cells in CD4⁺ T cells in the combination treatment group were significantly higher compared with the acute rejection group. Moreover, within the CD4⁺CD25⁺ T cell population the combination treatment recipients maintained a higher incidence of Foxp3⁺ T cells compared with the other groups. Despite the lower serum levels of interleukin (IL)‐2, IL‐12, and interferon‐γ in the combined treated group, the cytokine levels in the combined treated group at 7 days after OLT was nearly twice that at 3 days after OLT but decreased significantly compared with the other groups at 28 days after OLT. Serum IL‐10 level in the combined treated group was higher than the other groups. Conclusions: A single imDC infusion followed by a short postoperative course of Rapa prolongs liver allograft survival and enhances the expansion of Treg cells. This optimal protocol may be a promising administration protocol for the peritransplant tolerance induction.     

Intracellular Shigella remodels its LPS to dampen the innate immune recognition and evade inflammasome activation

LPS is a potent bacterial effector triggering the activation of the innate immune system following binding with the complex CD14, myeloid differentiation protein 2, and Toll-like receptor 4. The LPS of the enteropathogen Shigella flexneri is a hexa-acylated isoform possessing an optimal inflammatory activity. Symptoms of shigellosis are produced by severe inflammation caused by the invasion process of Shigella in colonic and rectal mucosa. Here we addressed the question of the role played by the Shigella LPS in eliciting a dysregulated inflammatory response of the host. We unveil that (i) Shigella is able to modify the LPS composition, e.g., the lipid A and core domains, during proliferation within epithelial cells; (ii) the LPS of intracellular bacteria (iLPS) and that of bacteria grown in laboratory medium differ in the number of acyl chains in lipid A, with iLPS being the hypoacylated; (iii) the immunopotential of iLPS is dramatically lower than that of bacteria grown in laboratory medium; (iv) both LPS forms mainly signal through the Toll-like receptor 4/myeloid differentiation primary response gene 88 pathway; (v) iLPS down-regulates the inflammasome-mediated release of IL-1β in Shigella-infected macrophages; and (vi) iLPS exhibits a reduced capacity to prime polymorfonuclear cells for an oxidative burst. We propose a working model whereby the two forms of LPS might govern different steps of the invasive process of Shigella. In the first phases, the bacteria, decorated with hypoacylated LPS, are able to lower the immune system surveillance, whereas, in the late phases, shigellae harboring immunopotent LPS are fully recognized by the immune system, which can then successfully resolve the infection.LPS is a glycolipid located in the outer membrane of Gram-negative bacteria. It is composed of three covalently linked domains: lipid A, which is embedded in the outer membrane; the oligosaccharide core; and the O-polysaccharide or O-antigen, which cover the bacterial surface. During infections sustained by Gram-negative bacteria, detection of LPS initiates an acute inflammatory response as LPS, mainly by the lipid A, which is the real pathogen-associated molecular pattern (PAMP), is sensed by the innate immune system, through the binding to the pattern recognition receptor (PRR) complex of myeloid differentiation protein 2 (MD-2) and Toll-like receptor (TLR) 4 (TLR4) (1–3). The downstream effects of LPS recognition elicit effector mechanisms aimed at pathogen eradication. However, LPS can also elicit an host reaction because it is a major mediator of pathologic processes (4). The strength of the innate immune response to LPS can be modulated by its chemical structure; specifically, a fine tuning of the lipid A structure can significantly affect the immunostimulatory properties of the whole LPS molecule (5, 6). There is a strong correlation between the number of acyl chains of lipid A and the immunological response via the TLR4 pathway. In general, hexaacylated lipid A species are agonists, whereas tetraacylated species are antagonists with a weak inflammatory potential (7). Gram-negative bacteria can synthesize a range of differentially acylated LPSs as a result of the LPS biosynthesis. Changes in lipid A acylation underlie the adaptation of pathogens to different hosts, such as Yersinia pestis (8), or to different phases of pathogenesis such as Salmonella typhimurium (9) or in the establishment of chronic infection such as Pseudomonas aeruginosa (10, 11).Shigella flexneri is a Gram-negative pathogen that infects humans. The ingestion of as few as 100 bacteria is sufficient to cause bacillary dysentery, a severe rectocolitis caused by the dramatic inflammatory reaction induced by Shigella invasion on the colonic and rectal mucosa (12). Shigella enters epithelial cells by injecting effectors via a type III secretion system (T3SS) (13), escapes from the phagocytic vacuole, and actively proliferates within the cytosol of infected cells (14, 15). Bacterial proliferation is a potent signal to initiate inflammation because intracellular shigellae activate NF-κB following recognition of peptidoglycan (PGN) by the PRR Nod1, leading to IL-8 production (16, 17). IL-8 attracts neutrophils that are required for the clearance of shigellae, but also participates in epithelial barrier destruction (18). In macrophages, Shigella is able to trigger the assembly of the inflammasome, an important defense mechanism that is part of the innate immune system (19). The inflammasome is a multiprotein complex that mediates activation of caspase-1, which promotes the secretion of the proinflammatory cytokines IL-1β and IL-18 as well as a cell death process called pyroptosis (20, 21). Different PRRs, i.e., TLRs and nucleotide-binding oligomerization domain-like receptors (NLRs) contribute to the inflammasome assembly (22). In Shigella-infected macrophages, the activation of the NLRC4-mediated inflammasome triggers cell death and release of IL-1β and IL-18 (19, 23). Indeed, production of IL-1β is a paradigm of shigellosis: the chief role of this cytokine has been highlighted in vivo in several studies (24–26).In tissues of animals and in ex vivo human samples infected with Shigella (27), a huge amount of LPS is usually observed, reflecting the presence of living bacteria and/or of processed molecules. However, whether, how, and at to what extent this mass of LPS present in Shigella-infected tissues could play a role in the inflammation remains largely unknown.In 2002, D’Hauteville et al. reported that, in S. flexneri, the lack of msbB genes, msbB1 and msbB2, both encoding the enzyme myristoyl transferase, reduces lipid A acylation degree along with TNF-α production and epithelial lining inflammatory destruction in a rabbit model of Shigella infection (28, 29). This study suggests that LPS composition can greatly influence the degree of inflammation induced by Shigella.In line with these issues, here we intend to contribute to the understanding of the role played by LPS in Shigella pathogenesis. Hence, we addressed the question of whether Shigella could adapt the LPS structure to the host thereby exploiting the mechanism of LPS modification to hijack the innate immune response. With this aim, we extracted, purified, and analyzed the LPS of shigellae resident in epithelial cells. We detailed the immunopotential of this structure and compared it to that of conventionally grown bacteria. Together our results point to a key role for LPS during the Shigella invasive process.We report that (i) Shigella is able to modify the LPS composition, e.g., the lipid A and core domains, during proliferation within epithelial cells; (ii) the LPS of intracellular bacteria (iLPS) and that of bacteria conventionally grown (aLPS) differ in the number of acyl chains in lipid A, with iLPS being hypoacylated; (iii) the immunopotential of iLPS is dramatically lower than that of aLPS; (iv) both LPS forms signal mainly through the TLR4/MyD88 pathway; (v) iLPS influences the inflammasome-mediated production of IL-1β in Shigella-infected macrophages; and (vi) iLPS exhibits a reduced capacity to prime PMNs for an oxidative burst.     

Translational shutdown and evasion of the innate immune response by SARS-CoV-2 NSP14 protein

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.