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2.
MBio ; 10(5)2019 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-31662460

RESUMO

E165R, a highly specific dUTP nucleotidohydrolase (dUTPase) encoded by the African swine fever virus (ASFV) genome, is required for productive replication of ASFV in swine macrophages. Here, we solved the high-resolution crystal structures of E165R in its apo state and in complex with its product dUMP. Structural analysis explicitly defined the architecture of the active site of the enzyme as well as the interaction between the active site and the dUMP ligand. By comparing the ASFV E165R structure with dUTPase structures from other species, we found that the active site of E165R is highly similar to those of dUTPases from Mycobacterium tuberculosis and Plasmodium falciparum, against which small-molecule chemicals have been developed, which could be the potential drug or lead compound candidates for ASFV. Our results provide important basis for anti-ASFV drug design by targeting E165R.IMPORTANCE African swine fever virus (ASFV), an Asfivirus affecting pigs and wild boars with up to 100% case fatality rate, is currently rampaging throughout China and some other countries in Asia. There is an urgent need to develop therapeutic and preventive reagents against the virus. Our crystallographic and biochemical studies reveal that ASFV E165R is a member of trimeric dUTP nucleotidohydrolase (dUTPase) family that catalyzes the hydrolysis of dUTP into dUMP. Our apo-E165R and E165R-dUMP structures reveal the constitutive residues and the configuration of the active center of this enzyme in rich detail and give evidence that the active center of E165R is very similar to that of dUTPases from Plasmodium falciparum and Mycobacterium tuberculosis, which have already been used as targets for designing drugs. Therefore, our high-resolution structures of E165R provide useful structural information for chemotherapeutic drug design.

3.
Emerg Infect Dis ; 25(11): 2136-2138, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31625865

RESUMO

We isolated Tamdy virus (TAMV; strain XJ01/TAMV/China/2018) from Hyalomma asiaticum ticks infesting Bactrian camels in Xinjiang, China, in 2018. The genome of the strain showed high nucleotide similarity with previously described TAMV strains from Asia. Our study highlights the potential threat of TAMV to public health in China.

4.
PLoS Biol ; 17(9): e3000436, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31498797

RESUMO

Bats harbor many zoonotic viruses, including highly pathogenic viruses of humans and other mammals, but they are typically asymptomatic in bats. To further understand the antiviral immunity of bats, we screened and identified a series of bat major histocompatibility complex (MHC) I Ptal-N*01:01-binding peptides derived from four different bat-borne viruses, i.e., Hendra virus (HeV), Ebola virus (EBOV), Middle East respiratory syndrome coronavirus (MERS-CoV), and H17N10 influenza-like virus. The structures of Ptal-N*01:01 display unusual peptide presentation features in that the bat-specific 3-amino acid (aa) insertion enables the tight "surface anchoring" of the P1-Asp in pocket A of bat MHC I. As the classical primary anchoring positions, the B and F pockets of Ptal-N*01:01 also show unconventional conformations, which contribute to unusual peptide motifs and distinct peptide presentation. Notably, the features of bat MHC I may be shared by MHC I from various marsupials. Our study sheds light on bat adaptive immunity and may benefit future vaccine development against bat-borne viruses of high impact on humans.

5.
Proc Natl Acad Sci U S A ; 116(38): 18928-18936, 2019 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-31467167

RESUMO

Prokaryotes possess CRISPR-Cas systems to exclude parasitic predators, such as phages and mobile genetic elements (MGEs). These predators, in turn, encode anti-CRISPR (Acr) proteins to evade the CRISPR-Cas immunity. Recently, AcrVA4, an Acr protein inhibiting the CRISPR-Cas12a system, was shown to diminish Lachnospiraceae bacterium Cas12a (LbCas12a)-mediated genome editing in human cells, but the underlying mechanisms remain elusive. Here we report the cryo-EM structures of AcrVA4 bound to CRISPR RNA (crRNA)-loaded LbCas12a and found AcrVA4 could inhibit LbCas12a at several stages of the CRISPR-Cas working pathway, different from other characterized type I/II Acr inhibitors which target only 1 stage. First, it locks the conformation of the LbCas12a-crRNA complex to prevent target DNA-crRNA hybridization. Second, it interacts with the LbCas12a-crRNA-dsDNA complex to release the bound DNA before cleavage. Third, AcrVA4 binds the postcleavage LbCas12a complex to possibly block enzyme recycling. These findings highlight the multifunctionality of AcrVA4 and provide clues for developing regulatory genome-editing tools.

6.
Front Immunol ; 10: 1709, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31396224

RESUMO

Human leukocyte antigen (HLA) alleles have a high degree of polymorphism, which determines their peptide-binding motifs and subsequent T-cell receptor recognition. The simplest way to understand the cross-presentation of peptides by different alleles is to classify these alleles into supertypes. A1 and A3 HLA supertypes are widely distributed in humans. However, direct structural and functional evidence for peptide presentation features of key alleles (e.g., HLA-A*30:01 and -A*30:03) are lacking. Herein, the molecular basis of peptide presentation of HLA-A*30:01 and -A*30:03 was demonstrated by crystal structure determination and thermostability measurements of complexes with T-cell epitopes from influenza virus (NP44), human immunodeficiency virus (RT313), and Mycobacterium tuberculosis (MTB). When binding to the HIV peptide, RT313, the PΩ-Lys anchoring modes of HLA-A*30:01, and -A*30:03 were similar to those of HLA-A*11:01 in the A3 supertype. However, HLA-A*30:03, but not -A*30:01, also showed binding with the HLA*01:01-favored peptide, NP44, but with a specific structural conformation. Thus, different from our previous understanding, HLA-A*30:01 and -A*30:03 have specific peptide-binding characteristics that may lead to their distinct supertype-featured binding peptide motifs. Moreover, we also found that residue 77 in the F pocket was one of the key residues for the divergent peptide presentation characteristics of HLA-A*30:01 and -A*30:03. Interchanging residue 77 between HLA-A*30:01 and HLA-A*30:03 switched their presented peptide profiles. Our results provide important recommendations for screening virus and tumor-specific peptides among the population with prevalent HLA supertypes for vaccine development and immune interventions.

8.
Emerg Microbes Infect ; 8(1): 1157-1167, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31373538

RESUMO

The spread of influenza A/H3N2 variants possessing the hemagglutinin 121 K mutation and the unexpectedly high incidence of influenza in the 2017-2018 northern hemisphere influenza season have raised serious concerns about the next pandemic. We summarized the national surveillance data of seasonal influenza in China and identified marked differences in influenza epidemics between northern and southern China, particularly the predominating subtype and the presence of an additional summer peak in southern China. Notably, a minor spring peak of influenza caused by a different virus subtype was also observed. We also revealed that the 3C.2a lineage was dominant from the summer of 2015 to the end of the 2015-2016 peak season in China, after which the 3C.2a2 lineage predominated despite the importation and co-circulation of the 121 K variants of 3C.2a1 and 3C.2a3 lineages at the global level. Finally, an analysis based on genetic distances revealed a delay in A/H3N2 vaccine strain update. Overall, our results highlight the complicated circulation pattern of seasonal influenza in China and the necessity for a timely vaccine strain update worldwide.

9.
Adv Healthc Mater ; 8(16): e1900456, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31267679

RESUMO

Influenza A virus (IAV), a deadly zoonotic pathogen, poses a tremendous threat and burden to global health systems. Pigs act as "mixing vessel" hosts to support and generate new pandemic viruses. Preventing the spread of IAV in pigs effectively can delay or even block cross-species transmission. Universal vaccines based on the highly conserved ectodomain of influenza matrix protein 2 (M2e) have been widely reported, but have not been applied due to inadequate protection. Porcine circovirus type 2 (PCV2) causes immunosuppression and promotes swine influenza virus (SIV) infection. Here, M2e is inserted into capsid protein of PCV2 without burying the neutralizing epitopes and self-assembles to form a bivalent nanovaccine. Inoculation with the nanovaccine induces robust M2e- and PCV2-specific immune responses. The nanovaccine confers protection against lethal challenges of IAV from different species in mice, and significantly reduces SIV titers in pigs' respiratory tract and blocks SIV transmission. These results indicate that the nanovaccine is an economical and promising PCV2 and universal IAV bivalent vaccine, and it will synergistically and powerfully offer potential ability to block IAV cross-species reassortment and transmission.

10.
Cell Mol Immunol ; 2019 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-31273318

RESUMO

Leukocyte immunoglobulin (Ig)-like receptors (LILRs), also known as CD85 and immunoglobulin-like transcripts (ILTs), play pivotal roles in regulating immune responses. These receptors define an immune checkpoint that immune therapy can target. Through cis or trans interactions with human leukocyte antigen (HLA)-G, the two most abundantly expressed inhibitory LILRs, LILRB1, and LILRB2 (LILRB1/2, also known as CD85j/d and ILT2/4), are involved in immunotolerance in pregnancy and transplantation, autoimmune diseases, and immune evasion by tumors. Although the discrete domains of LILRB1/2 are clear, the assembly mode of the four extracellular Ig-like domains (D1, D2, D3, and D4) remains unknown. Previous data indicate that D1D2 is responsible for binding to HLA class I (HLA-I), but the roles of D3D4 are still unclear. Here, we determined the crystal structure of the four Ig-like domain LILRB2 and four-domain LILRB1 in complex with HLA-G1. The angles between adjacent domains and the staggered assembly of the four domains suggest limited flexibility and limited plasticity of the receptors during ligand binding. The complex structure of four-domain LILRB1 and HLA-G1 supports the model that D1D2 is responsible for HLA-I binding, while D3D4 acts as a scaffold. Accordingly, cis and trans binding models for HLA-I binding to LILRB1/2 are proposed. The geometries of LILRB1/2 in complex with dimeric and monomeric HLA-G1 suggest the accessibility of the dimeric receptor, which in turn, transduces more inhibitory signals. The assembly of LILRB1/2 and its binding to HLA-G1 could aid in the design of immune regulators and benefit immune interference.

11.
Cancer Immunol Res ; 7(8): 1244-1257, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31213474

RESUMO

Therapeutic strategies are urgently needed for patients with acute myeloid leukemia (AML). Leukocyte immunoglobulin-like receptor B4 (LILRB4), which suppresses T-cell activation and supports tissue infiltration of AML cells, represents an attractive drug target for anti-AML therapeutics. Here, we report the identification and development of an LILRB4-specific humanized mAb that blocks LILRB4 activation. This mAb, h128-3, showed potent activity in blocking the development of monocytic AML in various models including patient-derived xenograft mice and syngeneic immunocompetent AML mice. MAb h128-3 enhanced the anti-AML efficacy of chemotherapy treatment by stimulating mobilization of leukemia cells. Mechanistic studies revealed four concordant modes of action for the anti-AML activity of h128-3: (i) reversal of T-cell suppression, (ii) inhibition of monocytic AML cell tissue infiltration, (iii) antibody-dependent cellular cytotoxicity, and (iv) antibody-dependent cellular phagocytosis. Therefore, targeting LILRB4 with antibody represents an effective therapeutic strategy for treating monocytic AML.

12.
Mol Immunol ; 112: 274-282, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31226552

RESUMO

The viral peptides presentation by major histocompatibility complex class I (MHC I) molecules play a pivotal role in T-cell recognition and the subsequent virus clearance. This process is delicately adjusted by the variant residues of MHC I, especially the residues in the peptide binding groove (PBG). In a series of MHC I molecules, a salt bridge is formed above the N-terminus of the peptides. However, the potential impact of the salt bridge on peptide binding and T-cell receptor (TCR) recognition of MHC I, as well as the corresponding molecular basis, are still largely unknown. Herein, we determined the structures of HLA-B*4001 and H-2Kd in which two different types of salt bridges (Arg62-Glu163 or Arg66-Glu163) across the PBG were observed. Although the two salt bridges led to different conformation shifts of both the MHC I α helix and the peptides, binding of the peptides with the salt bridge residues was relatively conserved. Furthermore, through a series of in vitro and in vivo investigations, we found that MHC I mutations that disrupt the salt bridge alleviate peptide binding and can weaken the TCR recognition of MHC I-peptide complexes. Our study may provide key references for understanding MHC I-restricted peptide recognition by T-cells.


Assuntos
Apresentação do Antígeno/imunologia , Genes MHC Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Peptídeos/imunologia , Ligação Proteica/imunologia , Linfócitos T/imunologia , Animais , Sítios de Ligação/imunologia , Feminino , Antígenos HLA-B/imunologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Conformação Proteica , Receptores de Antígenos de Linfócitos T/imunologia
13.
Nat Microbiol ; 4(10): 1750-1759, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31209309

RESUMO

The influenza virus polymerase uses capped RNA primers to initiate transcription, and a combination of terminal and internal de novo initiations for the two-step replication process by binding the conserved viral genomic RNA (vRNA) or complementary RNA (cRNA) promoter. Here, we determined the apo and promoter-bound influenza D polymerase structures using cryo-electron microscopy and found the polymerase has an evolutionarily conserved stable core structure with inherently flexible peripheral domains. Strikingly, two conformations (mode A and B) of the vRNA promoter were observed where the 3'-vRNA end can bind at two different sites, whereas the cRNA promoter only binds in the mode B conformation. Functional studies confirmed the critical role of the mode B conformation for vRNA synthesis via the intermediate cRNA but not for cRNA production, which is mainly regulated by the mode A conformation. Both conformations participate in the regulation of the transcription process. This work advances our understanding of the regulatory mechanisms for the synthesis of different RNA species by influenza virus polymerase and opens new opportunities for antiviral drug design.

14.
Euro Surveill ; 24(21)2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31138362

RESUMO

After no reported human cases of highly pathogenic avian influenza (HPAI) H7N9 for over a year, a case with severe disease occurred in late March 2019. Among HPAI H7N9 viral sequences, those recovered from the case and from environmental samples of a poultry slaughtering stall near their home formed a distinct clade from 2017 viral sequences. Several mutations possibly associated to antigenic drift occurred in the haemagglutinin gene, potentially warranting update of H7N9 vaccine strains.

15.
Cell ; 177(7): 1714-1724.e12, 2019 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-31080063

RESUMO

Arthritogenic alphaviruses, such as Chikungunya virus (CHIKV), cause severe and debilitating rheumatic diseases worldwide, resulting in severe morbidity and economic costs. Recently, MXRA8 was reported as an entry receptor. Here, we present the crystal structures of the mouse MXRA8, human MXRA8 in complex with the CHIKV E protein, and the cryo-electron microscopy structure of human MXRA8 and CHIKV virus-like particle. MXRA8 has two Ig-like domains with unique structural topologies. This receptor binds in the "canyon" between two protomers of the E spike on the surface of the virion. The atomic details at the interface between the two binding entities reveal that both the two domains and the hinge region of MXRA8 are involved in interaction with CHIKV E1-E2 residues from two protomers. Notably, the stalk region of MXRA8 is critical for CHIKV virus entry. This finding provides important information regarding the development of therapeutic countermeasures against those arthritogenic alphaviruses.

16.
Cell ; 177(5): 1086-1088, 2019 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-31100263

RESUMO

A universal vaccine against influenza remains a critical target, and efforts have recently focused on the stem of the hemagglutinin glycoprotein. In this issue of Cell and a related Cell Host & Microbe article, three studies identify broad protective epitopes in the hemagglutinin head domain that are exposed by trimer "breathing."

17.
Cell ; 177(6): 1553-1565.e16, 2019 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-31104841

RESUMO

Enterovirus B (EV-B), a major proportion of the genus Enterovirus in the family Picornaviridae, is the causative agent of severe human infectious diseases. Although cellular receptors for coxsackievirus B in EV-B have been identified, receptors mediating virus entry, especially the uncoating process of echovirus and other EV-B remain obscure. Here, we found that human neonatal Fc receptor (FcRn) is the uncoating receptor for major EV-B. FcRn binds to the virus particles in the "canyon" through its FCGRT subunit. By obtaining multiple cryo-electron microscopy structures at different stages of virus entry at atomic or near-atomic resolution, we deciphered the underlying mechanisms of enterovirus attachment and uncoating. These structures revealed that different from the attachment receptor CD55, binding of FcRn to the virions induces efficient release of "pocket factor" under acidic conditions and initiates the conformational changes in viral particle, providing a structural basis for understanding the mechanisms of enterovirus entry.

18.
Nat Microbiol ; 4(7): 1231-1241, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30936489

RESUMO

Rift Valley fever virus (RVFV) is a mosquito-borne pathogen that causes substantial morbidity and mortality in livestock and humans. To date, there are no licensed human vaccines or therapeutics available. Here, we report the isolation of monoclonal antibodies from a convalescent patient, targeting the RVFV envelope proteins Gn and Gc. The Gn-specific monoclonal antibodies exhibited much higher neutralizing activities in vitro and protection efficacies in mice against RVFV infection, compared to the Gc-specific monoclonal antibodies. The Gn monoclonal antibodies were found to interfere with soluble Gn binding to cells and prevent infection by blocking the attachment of virions to host cells. Structural analysis of Gn complexed with four Gn-specific monoclonal antibodies resulted in the definition of three antigenic patches (A, B and C) on Gn domain I. Both patches A and B are major neutralizing epitopes. Our results highlight the potential of antibody-based therapeutics and provide a structure-based rationale for designing vaccines against RVFV.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Febre do Vale de Rift/prevenção & controle , Vírus da Febre do Vale do Rift/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Antivirais/administração & dosagem , Cercopithecus aethiops , Cristalografia por Raios X , Epitopos , Feminino , Humanos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Domínios Proteicos , Febre do Vale de Rift/imunologia , Células Vero , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/metabolismo , Ligação Viral
19.
iScience ; 14: 113-124, 2019 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-30952089

RESUMO

Programmed cell death 1 (PD-1)/PD-1 ligand-1 (PD-L1)-blocking monoclonal antibodies (mAbs) have taken center stage for tumor immune checkpoint therapy. Identification of the "hotspots" on PD-1 for mAbs will help to develop next-generation oral deliverable agents with long-lasting efficacy. Here, we identified two PD-1-targeting mAbs, GY-5 and GY-14, with PD-1/PD-L1-blocking efficacy. Complex structural information revealed that both mAbs mainly bind to the FG loop of PD-1, which also contributes multiple interactions with PD-L1. The FG loop adopts substantially varied conformations upon binding to different mAbs, providing a novel targetable region for the development of PD-1-specific biologics and small chemical molecules. Glycosylation modifications of PD-1 could be observed in three of the four potential N-linked glycosylation sites. However, the binding of GY-5 and GY-14 to PD-1 was not affected by glycosylation. These findings broaden our understanding of the mechanism of anti-PD-1 mAbs and provide insight into the development of agents targeting PD-1.

20.
Cell Rep ; 27(4): 1205-1220.e4, 2019 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-31018134

RESUMO

Regulatory T cell (Treg) activation is crucial for maintaining self-tolerance, but the translational regulation of this process is still poorly understood. Although ribosome biogenesis is considered a housekeeping process, emerging evidence supports the hypothesis that ribosome biogenesis can selectively regulate protein synthesis by tuning translation. Here, we focused on the ribosome biogenesis factor Noc4L, based on the observations that Noc4L is highly expressed in activated Tregs. Conditional Noc4L knockout in Tregs resulted in a lethal autoimmune phenotype resembling Treg-deficient scurfy mice. Interestingly, the Noc4L defect did not globally affect overall protein translation in Tregs but was selectively detrimental to the expression of mRNAs related to Treg activation. These results demonstrate the critical role of Noc4L-mediated ribosome biogenesis in controlling the activation of Tregs and maintaining immune tolerance.

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