RESUMO
SARS-CoV-2, the causative agent of COVID-19, has been intensely studied in search of effective antiviral treatments. The immunosuppressant cyclosporine A (CsA) has been suggested to be a pan-coronavirus inhibitor, yet its underlying mechanism remained largely unknown. Here, we found that non-structural protein 1 (Nsp1) of SARS-CoV-2 usurped CsA-suppressed nuclear factor of activated T cells (NFAT) signaling to drive the expression of cellular DEAD-box helicase 5 (DDX5), which facilitates viral replication. Nsp1 interacted with calcineurin A (CnA) to displace the regulatory protein regulator of calcineurin 3 (RCAN3) of CnA for NFAT activation. The influence of NFAT activation on SARS-CoV-2 replication was also validated by using the Nsp1-deficient mutant virus. Calcineurin inhibitors, such as CsA and VIVIT, inhibited SARS-CoV-2 replication and exhibited synergistic antiviral effects when used in combination with nirmatrelvir. Our study delineated the molecular mechanism of CsA-mediated inhibition of SARS-CoV-2 replication and the anti-SARS-CoV-2 action of calcineurin inhibitors. IMPORTANCE: Cyclosporine A (CsA), commonly used to inhibit immune responses, is also known to have anti-SARS-CoV-2 activity, but its mode of action remains elusive. Here, we provide a model to explain how CsA antagonizes SARS-CoV-2 through three critical proteins: DDX5, NFAT1, and Nsp1. DDX5 is a cellular facilitator of SARS-CoV-2 replication, and NFAT1 controls the production of DDX5. Nsp1 is a viral protein absent from the mature viral particle and capable of activating the function of NFAT1 and DDX5. CsA and similar agents suppress Nsp1, NFAT1, and DDX5 to exert their anti-SARS-CoV-2 activity either alone or in combination with Paxlovid.
Assuntos
COVID-19 , SARS-CoV-2 , Transdução de Sinais , Proteínas não Estruturais Virais , Humanos , Antivirais , Calcineurina/metabolismo , Inibidores de Calcineurina/farmacologia , COVID-19/virologia , Ciclosporina/farmacologia , Fatores de Transcrição NFATC/metabolismo , SARS-CoV-2/fisiologia , Proteínas não Estruturais Virais/metabolismoRESUMO
Epstein-Barr virus (EBV) has developed effective strategies to evade host innate immune responses. Here we reported on mitigation of type I interferon (IFN) production by EBV deubiquitinase (DUB) BPLF1 through cGAS-STING and RIG-I-MAVS pathways. The two naturally occurring forms of BPLF1 exerted potent suppressive effect on cGAS-STING-, RIG-I- and TBK1-induced IFN production. The observed suppression was reversed when DUB domain of BPLF1 was rendered catalytically inactive. The DUB activity of BPLF1 also facilitated EBV infection by counteracting cGAS-STING- and TBK1-mediated antiviral defense. BPLF1 associated with STING to act as an effective DUB targeting its K63-, K48- and K27-linked ubiquitin moieties. BPLF1 also catalyzed removal of K63- and K48-linked ubiquitin chains on TBK1 kinase. The DUB activity of BPLF1 was required for its suppression of TBK1-induced IRF3 dimerization. Importantly, in cells stably carrying EBV genome that encodes a catalytically inactive BPLF1, the virus failed to suppress type I IFN production upon activation of cGAS and STING. This study demonstrated IFN antagonism of BPLF1 mediated through DUB-dependent deubiquitination of STING and TBK1 leading to suppression of cGAS-STING and RIG-I-MAVS signaling.
Assuntos
Infecções por Vírus Epstein-Barr , Herpesvirus Humano 4 , Humanos , Proteína DEAD-box 58 , Enzimas Desubiquitinantes , Infecções por Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/metabolismo , Imunidade Inata , Nucleotidiltransferases/metabolismo , UbiquitinaRESUMO
Single-cycle infectious virus can elicit close-to-natural immune response and memory. One approach to generate single-cycle severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is through deletion of structural genes such as spike (S) and nucleocapsid (N). Transcomplementation of the resulting ΔS or ΔN virus through enforced expression of S or N protein in the cells gives rise to a live but unproductive virus. In this study, ΔS and ΔN BAC clones were constructed and their live virions were rescued by transient expression of S and N proteins from the ancestral and the Omicron strains. ΔS and ΔN virions were visualized by transmission electron microscopy. Virion production of ΔS was more efficient than that of ΔN. The coated S protein from ΔS was delivered to infected cells in which the expression of N protein was also robust. In contrast, expression of neither S nor N was detected in ΔN-infected cells. ΔS underwent viral RNA replication, induced type I interferon (IFN) response, but did not form plaques. Despite RNA replication in cells, ΔS infection did not produce viral progeny in culture supernatant. Interestingly, viral RNA replication was not further enhanced upon overexpression of S protein. Taken together, our work provides a versatile platform for development of single-cycle vaccines for SARS-CoV-2.
Assuntos
COVID-19 , Interferon Tipo I , Vacinas contra COVID-19 , Humanos , Interferon Tipo I/genética , RNA Viral/genética , Replicon , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genéticaRESUMO
Rapid development and successful use of vaccines against SARS-CoV-2 might hold the key to curb the ongoing pandemic of COVID-19. Emergence of vaccine-evasive SARS-CoV-2 variants of concern (VOCs) has posed a new challenge to vaccine design and development. One urgent need is to determine what types of variant-specific and bivalent vaccines should be developed. Here, we compared homotypic and heterotypic protection against SARS-CoV-2 infection of hamsters with monovalent and bivalent whole-virion inactivated vaccines derived from representative VOCs. In addition to the ancestral SARS-CoV-2 Wuhan strain, Delta (B.1.617.2; δ) and Theta (P.3; θ) variants were used in vaccine preparation. Additional VOCs including Omicron (B.1.1.529) and Alpha (B.1.1.7) variants were employed in the challenge experiment. Consistent with previous findings, Omicron variant exhibited the highest degree of immune evasion, rendering all different forms of inactivated vaccines substantially less efficacious. Notably, monovalent and bivalent Delta variant-specific inactivated vaccines provided optimal protection against challenge with Delta variant. Yet, some cross-variant protection against Omicron and Alpha variants was seen with all monovalent and bivalent inactivated vaccines tested. Taken together, our findings support the notion that an optimal next-generation inactivated vaccine against SARS-CoV-2 should contain the predominant VOC in circulation. Further investigations are underway to test whether a bivalent vaccine for Delta and Omicron variants can serve this purpose.
Assuntos
COVID-19 , Vacinas Virais , Animais , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Cricetinae , Humanos , SARS-CoV-2 , Vacinas Combinadas , Vacinas de Produtos InativadosRESUMO
Some lytic proteins encoded by Epstein-Barr virus (EBV) suppress host interferon (IFN) signaling to facilitate viral replication. In this study, we sought to identify and characterize EBV proteins antagonizing IFN signaling. The induction of IFN-stimulated genes (ISGs) by IFN-ß was effectively suppressed by EBV. A functional screen was therefore performed to identify IFN-antagonizing proteins encoded by EBV. EBV tegument protein BGLF2 was identified as a potent suppressor of JAK-STAT signaling. This activity was found to be independent of its stimulatory effect on p38 and JNK pathways. Association of BGLF2 with STAT2 resulted in more pronounced K48-linked polyubiquitination and proteasomal degradation of the latter. Mechanistically, BGLF2 promoted the recruitment of SHP1 phosphatase to STAT1 to inhibit its tyrosine phosphorylation. In addition, BGLF2 associated with cullin 1 E3 ubiquitin ligase to facilitate its recruitment to STAT2. Consequently, BGLF2 suppressed ISG induction by IFN-ß. Furthermore, BGLF2 also suppressed type II and type III IFN signaling, although the suppressive effect on type II IFN response was milder. When pretreated with IFN-ß, host cells became less susceptible to primary infection of EBV. This phenotype was reversed when expression of BGLF2 was enforced. Finally, genetic disruption of BGLF2 in EBV led to more pronounced induction of ISGs. Our study unveils the roles of BGLF2 not only in the subversion of innate IFN response but also in lytic infection and reactivation of EBV. IMPORTANCE Epstein-Barr virus (EBV) is an oncogenic virus associated with the development of lymphoid and epithelial malignancies. EBV has to subvert interferon-mediated host antiviral response to replicate and cause diseases. It is therefore of great interest to identify and characterize interferon-antagonizing proteins produced by EBV. In this study, we perform a screen to search for EBV proteins that suppress the action of interferons. We further show that BGLF2 protein of EBV is particularly strong in this suppression. This is achieved by inhibiting two key proteins STAT1 and STAT2 that mediate the antiviral activity of interferons. BGLF2 recruits a host enzyme to remove the phosphate group from STAT1 thereby inactivating its activity. BGLF2 also redirects STAT2 for degradation. A recombinant virus in which BGLF2 gene has been disrupted can activate host interferon response more robustly. Our findings reveal a novel mechanism by which EBV BGLF2 protein suppresses interferon signaling.
Assuntos
Infecções por Vírus Epstein-Barr/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Proteínas Virais de Fusão/metabolismo , Herpesvirus Humano 4/metabolismo , Interações Hospedeiro-Patógeno , Interferons/metabolismo , Sistema de Sinalização das MAP Quinases , Fosforilação , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT2/metabolismo , Transdução de Sinais , Proteínas Virais de Fusão/genética , Replicação ViralRESUMO
Epstein-Barr virus (EBV) infects more than 90% of the world's adult population. Once established, latent infection of nasopharyngeal epithelial cells with EBV is difficult to eradicate and might lead to the development of nasopharyngeal carcinoma (NPC) in a small subset of individuals. In this study we explored the anti-EBV potential of CRISPR/Cas9 targeting of EBV genome in infected NPC cells. We designed gRNAs to target different regions of the EBV genome and transfected them into C666-1 cells. The levels of EBV DNA in transfected cells were decreased by about 50%. The suppressive effect on EBV DNA load lasted for weeks but could not be further enhanced by re-transfection of gRNA. Suppression of EBV by CRISPR/Cas9 did not affect survival of C666-1 cells but sensitized them to chemotherapeutic killing by cisplatin and 5-fluorouracil. Our work provides the proof-of-principle for suppressing EBV DNA load with CRISPR/Cas9 and a potential new strategy to sensitize EBV-infected NPC cells to chemotherapy.