RESUMO
Alkaline exonucleases (AE) are present in several large DNA viruses including bacteriophage λ and herpesviruses, where they play roles in viral DNA processing during genome replication. Given the genetic conservation of AEs across viruses infecting different kingdoms of life, these enzymes likely assume central roles in the lifecycles of viruses where they have yet to be well characterized. Here, we applied a structure-guided functional analysis of the bifunctional AE in the oncogenic human gammaherpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV), called SOX. In addition to identifying a preferred DNA substrate preference for SOX, we define key residues important for DNA binding and DNA processing, and how SOX activity on DNA partially overlaps with its functionally separable cleavage of mRNA. By engineering these SOX mutants into KSHV, we reveal roles for its DNase activity in viral gene expression and infectious virion production. Our results provide mechanistic insight into gammaherpesviral AE activity as well as areas of functional conservation between this mammalian virus AE and its distant relative in phage λ.
Assuntos
Exonucleases , Herpesvirus Humano 8 , Animais , Humanos , DNA Viral/metabolismo , Exonucleases/genética , Expressão Gênica , Regulação Viral da Expressão Gênica , Herpesvirus Humano 8/metabolismo , Mamíferos/genética , Vírion/metabolismo , Replicação ViralRESUMO
A number of viruses remodel the cellular gene expression landscape by globally accelerating messenger RNA (mRNA) degradation. Unlike the mammalian basal mRNA decay enzymes, which largely target mRNA from the 5' and 3' end, viruses instead use endonucleases that cleave their targets internally. This is hypothesized to more rapidly inactivate mRNA while maintaining selective power, potentially though the use of a targeting motif(s). Yet, how mRNA endonuclease specificity is achieved in mammalian cells remains largely unresolved. Here, we reveal key features underlying the biochemical mechanism of target recognition and cleavage by the SOX endonuclease encoded by Kaposi's sarcoma-associated herpesvirus (KSHV). Using purified KSHV SOX protein, we reconstituted the cleavage reaction in vitro and reveal that SOX displays robust, sequence-specific RNA binding to residues proximal to the cleavage site, which must be presented in a particular structural context. The strength of SOX binding dictates cleavage efficiency, providing an explanation for the breadth of mRNA susceptibility observed in cells. Importantly, we establish that cleavage site specificity does not require additional cellular cofactors, as had been previously proposed. Thus, viral endonucleases may use a combination of RNA sequence and structure to capture a broad set of mRNA targets while still preserving selectivity.
Assuntos
Endonucleases/genética , Herpesvirus Humano 8/enzimologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas com Domínio LIM/metabolismo , Proteínas Virais/genética , Animais , Sítios de Ligação , Endonucleases/metabolismo , Regulação Viral da Expressão Gênica , Herpesvirus Humano 8/genética , Humanos , Insetos , Cinética , Mutação , Clivagem do RNA , Estabilidade de RNA/genética , RNA Mensageiro/metabolismo , Especificidade por Substrato , Proteínas Virais/metabolismoRESUMO
The kinase/endonuclease IRE1 is the most conserved signal transducer of the unfolded protein response (UPR), an intracellular signaling network that monitors and regulates the protein folding capacity of the endoplasmic reticulum (ER). Upon sensing protein folding perturbations in the ER, IRE1 initiates the unconventional splicing of XBP1 mRNA culminating in the production of the transcription factor XBP1s, which expands the ER's protein folding capacity. We show that an RNA-intrinsic conformational change causes the intron of XBP1 mRNA to be ejected and the exons to zipper up into an extended stem, juxtaposing the RNA ends for ligation. These conformational rearrangements are important for XBP1 mRNA splicing in vivo. The features that point to such active participation of XBP1 mRNA in the splicing reaction are highly conserved throughout metazoan evolution, supporting their importance in orchestrating XBP1 mRNA processing with efficiency and fidelity.
Assuntos
Proteínas de Ligação a DNA/genética , Íntrons , Splicing de RNA , Fatores de Transcrição/genética , Animais , Sequência de Bases , Sequência Conservada , Proteínas de Ligação a DNA/metabolismo , Retículo Endoplasmático/genética , Endorribonucleases/genética , Endorribonucleases/metabolismo , Humanos , Conformação de Ácido Nucleico , Dobramento de Proteína , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro , Fatores de Transcrição de Fator Regulador X , Alinhamento de Sequência , Análise de Sequência de DNA , Fatores de Transcrição/metabolismo , Transcrição Gênica , Resposta a Proteínas não Dobradas , Proteína 1 de Ligação a X-BoxRESUMO
Membrane proteins comprise a third of the human genome, yet present challenging targets for reverse chemical genetics. For example, although implicated in numerous diseases including multiple myeloma, the membrane protein caveolin-1 appears to offer a poor target for the discovery of synthetic ligands due to its largely unknown structure and insolubility. To break this impasse and identify new classes of caveolae controlling lead compounds, we applied phage-based, reverse chemical genetics for the discovery of caveolin-1 ligands derived from the anti-HIV therapeutic T20. Substitution of homologous residues into the T20 sequence used a process analogous to medicinal chemistry for the affinity maturation to bind caveolin. The resultant caveolin-1 ligands bound with >1000-fold higher affinity than wild-type T20. Two types of ELISAs and isothermal titration calorimetry (ITC) measurements demonstrated high affinity binding to caveolin by the T20 variants with K(d) values in the 150 nM range. Microscopy experiments with the highest affinity caveolin ligands confirmed colocalization of the ligands with endogenous caveolin in NIH 3T3 cells. The results establish the foundation for targeting caveolin and caveolae formation in living cells.
Assuntos
Caveolina 1/metabolismo , Evolução Molecular Direcionada/métodos , Descoberta de Drogas/métodos , Células 3T3 , Animais , Cavéolas , Caveolina 1/antagonistas & inibidores , Enfuvirtida , Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/farmacologia , Inibidores da Fusão de HIV , Humanos , Ligantes , Proteínas de Membrana , Camundongos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Ligação ProteicaRESUMO
Nonstructural protein 1 (nsp1) is a coronavirus (CoV) virulence factor that restricts cellular gene expression by inhibiting translation through blocking the mRNA entry channel of the 40S ribosomal subunit and by promoting mRNA degradation. We perform a detailed structure-guided mutational analysis of severe acute respiratory syndrome (SARS)-CoV-2 nsp1, revealing insights into how it coordinates these activities against host but not viral mRNA. We find that residues in the N-terminal and central regions of nsp1 not involved in docking into the 40S mRNA entry channel nonetheless stabilize its association with the ribosome and mRNA, both enhancing its restriction of host gene expression and enabling mRNA containing the SARS-CoV-2 leader sequence to escape translational repression. These data support a model in which viral mRNA binding functionally alters the association of nsp1 with the ribosome, which has implications for drug targeting and understanding how engineered or emerging mutations in SARS-CoV-2 nsp1 could attenuate the virus.
Assuntos
COVID-19/genética , Regulação Viral da Expressão Gênica , SARS-CoV-2/genética , Proteínas não Estruturais Virais/metabolismo , Anisotropia , COVID-19/imunologia , Análise Mutacional de DNA , Feminino , Perfilação da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Cinética , Mutação , Fenótipo , Mutação Puntual , Biossíntese de Proteínas , Domínios Proteicos , Estabilidade de RNA , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Ribossomos/metabolismoRESUMO
Phage display of protein and peptide libraries offers a powerful technology for the selection and isolation of ligands and receptors. To date, the technique has been considered limited to soluble, non-membrane proteins. We report two examples of phage display of full-length, folded and functional membrane proteins. Consistent display required the recently reported KO7(+) helper phage. The two proteins, full-length caveolin-1 and HIV gp41, display well on the surface of the phage, and maintain their binding activities as shown by in vitro assays.
Assuntos
Proteínas de Membrana/química , Modelos Moleculares , Proteínas da Matriz Viral/química , Bacteriófagos/química , Caveolina 1/química , Caveolina 1/metabolismo , Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/metabolismo , Humanos , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Biblioteca de Peptídeos , Relação Estrutura-Atividade , Proteínas da Matriz Viral/imunologia , Proteínas da Matriz Viral/metabolismoRESUMO
Two ER membrane-resident transmembrane kinases, IRE1 and PERK, function as stress sensors in the unfolded protein response. IRE1 also has an endoribonuclease activity, which initiates a non-conventional mRNA splicing reaction, while PERK phosphorylates eIF2α. We engineered a potent small molecule, IPA, that binds to IRE1's ATP-binding pocket and predisposes the kinase domain to oligomerization, activating its RNase. IPA also inhibits PERK but, paradoxically, activates it at low concentrations, resulting in a bell-shaped activation profile. We reconstituted IPA-activation of PERK-mediated eIF2α phosphorylation from purified components. We estimate that under conditions of maximal activation less than 15% of PERK molecules in the reaction are occupied by IPA. We propose that IPA binding biases the PERK kinase towards its active conformation, which trans-activates apo-PERK molecules. The mechanism by which partial occupancy with an inhibitor can activate kinases may be wide-spread and carries major implications for design and therapeutic application of kinase inhibitors.
Assuntos
Trifosfato de Adenosina/farmacologia , Endorribonucleases/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Resposta a Proteínas não Dobradas/efeitos dos fármacos , eIF-2 Quinase/antagonistas & inibidores , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/síntese química , Animais , Bioensaio , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Estresse do Retículo Endoplasmático , Endorribonucleases/genética , Endorribonucleases/metabolismo , Ativação Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Expressão Gênica , Genes Reporter , Células HEK293 , Humanos , Camundongos , Mimetismo Molecular , Inibidores de Proteínas Quinases/síntese química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição de Fator Regulador X , Radioisótopos de Enxofre , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Resposta a Proteínas não Dobradas/genética , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismoRESUMO
The general translation initiation factor eIF2 is a major translational control point. Multiple signaling pathways in the integrated stress response phosphorylate eIF2 serine-51, inhibiting nucleotide exchange by eIF2B. ISRIB, a potent drug-like small molecule, renders cells insensitive to eIF2α phosphorylation and enhances cognitive function in rodents by blocking long-term depression. ISRIB was identified in a phenotypic cell-based screen, and its mechanism of action remained unknown. We now report that ISRIB is an activator of eIF2B. Our reporter-based shRNA screen revealed an eIF2B requirement for ISRIB activity. Our results define ISRIB as a symmetric molecule, show ISRIB-mediated stabilization of activated eIF2B dimers, and suggest that eIF2B4 (δ-subunit) contributes to the ISRIB binding site. We also developed new ISRIB analogs, improving its EC50 to 600 pM in cell culture. By modulating eIF2B function, ISRIB promises to be an invaluable tool in proof-of-principle studies aiming to ameliorate cognitive defects resulting from neurodegenerative diseases.
Assuntos
Acetamidas/química , Cicloexilaminas/química , Fator de Iniciação 2B em Eucariotos/genética , Fármacos Neuroprotetores/química , Nootrópicos/química , Subunidades Proteicas/genética , Acetamidas/síntese química , Acetamidas/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cicloexilaminas/síntese química , Cicloexilaminas/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Fator de Iniciação 2B em Eucariotos/antagonistas & inibidores , Fator de Iniciação 2B em Eucariotos/metabolismo , Expressão Gênica , Genes Reporter , Células HEK293 , Células HeLa , Ensaios de Triagem em Larga Escala , Humanos , Células K562 , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Fármacos Neuroprotetores/síntese química , Fármacos Neuroprotetores/farmacologia , Nootrópicos/síntese química , Nootrópicos/farmacologia , Fosforilação , Ligação Proteica , Multimerização Proteica/efeitos dos fármacos , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Relação Estrutura-Atividade , Tapsigargina/antagonistas & inibidores , Tapsigargina/farmacologiaRESUMO
Protein folding by the endoplasmic reticulum (ER) is physiologically critical; its disruption causes ER stress and augments disease. ER stress activates the unfolded protein response (UPR) to restore homeostasis. If stress persists, the UPR induces apoptotic cell death, but the mechanisms remain elusive. Here, we report that unmitigated ER stress promoted apoptosis through cell-autonomous, UPR-controlled activation of death receptor 5 (DR5). ER stressors induced DR5 transcription via the UPR mediator CHOP; however, the UPR sensor IRE1α transiently catalyzed DR5 mRNA decay, which allowed time for adaptation. Persistent ER stress built up intracellular DR5 protein, driving ligand-independent DR5 activation and apoptosis engagement via caspase-8. Thus, DR5 integrates opposing UPR signals to couple ER stress and apoptotic cell fate.