RESUMO
Cytosolic long dsRNA, among the most potent proinflammatory signals, is recognized by melanoma differentiation-associated protein 5 (MDA5). MDA5 binds dsRNA cooperatively forming helical filaments. ATP hydrolysis by MDA5 fulfills a proofreading function by promoting dissociation of shorter endogenous dsRNs from MDA5 while allowing longer viral dsRNAs to remain bound leading to activation of interferon-ß responses. Here, we show that adjacent MDA5 subunits in MDA5-dsRNA filaments hydrolyze ATP cooperatively, inducing cooperative filament disassembly. Consecutive rounds of ATP hydrolysis amplify the filament footprint, displacing tightly bound proteins from dsRNA. Our electron microscopy and biochemical assays show that LGP2 binds to dsRNA at internal binding sites through noncooperative ATP hydrolysis. Unlike MDA5, LGP2 has low nucleic acid selectivity and can hydrolyze GTP and CTP as well as ATP. Binding of LGP2 to dsRNA promotes nucleation of MDA5 filament assembly resulting in shorter filaments. Molecular modeling identifies an internally bound MDA5-LGP2-RNA complex, with the LGP2 C-terminal tail forming the key contacts with MDA5. These contacts are specifically required for NTP-dependent internal RNA binding. We conclude that NTPase-dependent binding of LGP2 to internal dsRNA sites complements NTPase-independent binding to dsRNA ends, via distinct binding modes, to increase the number and signaling output of MDA5-dsRNA complexes.
Assuntos
RNA Helicases DEAD-box , Helicase IFIH1 Induzida por Interferon , RNA Helicases , RNA de Cadeia Dupla , RNA Viral , Trifosfato de Adenosina/metabolismo , RNA Helicases DEAD-box/metabolismo , Hidrólise , Imunidade Inata , Helicase IFIH1 Induzida por Interferon/genética , Helicase IFIH1 Induzida por Interferon/metabolismo , Nucleosídeo-Trifosfatase/genética , Nucleosídeo-Trifosfatase/metabolismo , RNA Helicases/metabolismo , RNA de Cadeia Dupla/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , HumanosRESUMO
Nonstructural protein 13 (nsp13), the helicase of SARS-CoV-2, has been shown to possess multiple functions that are essential for viral replication, and is considered an attractive target for the development of novel antivirals. We were initially interested in the interplay between nsp13 and interferon (IFN) signaling, and found that nsp13 inhibited reporter signal in an IFN-ß promoter assay. Surprisingly, the ectopic expression of different components of the RIG-I/MDA5 pathway, which were used to stimulate IFN-ß promoter, was also mitigated by nsp13. However, endogenous expression of these genes was not affected by nsp13. Interestingly, nsp13 restricted the expression of foreign genes originating from plasmid transfection, but failed to inhibit them after chromosome integration. These data, together with results from a runoff transcription assay and RNA sequencing, suggested a specific inhibition of episomal but not chromosomal gene transcription by nsp13. By using different truncated and mutant forms of nsp13, we demonstrated that its NTPase and helicase activities contributed to the inhibition of episomal DNA transcription, and that this restriction required direct interaction with episomal DNA. Based on these findings, we developed an economical and convenient high-throughput drug screening method targeting nsp13. We evaluated the inhibitory effects of various compounds on nsp13 by the expression of reporter gene plasmid after co-transfection with nsp13. In conclusion, we found that nsp13 can specifically inhibit episomal DNA transcription and developed a high-throughput drug screening method targeting nsp13 to facilitate the development of new antiviral drugs. IMPORTANCE To combat COVID-19, we need to understand SARS-CoV-2 and develop effective antiviral drugs. In our study, we serendipitously found that SARS-CoV-2 nsp13 could suppress episomal DNA transcription without affecting chromosomal DNA. Detailed characterization revealed that nsp13 suppresses episomal gene expression through its NTPase and helicase functions following DNA binding. Furthermore, we developed a high-throughput drug screening system targeting SARS-CoV-2 nsp13. Compared to traditional SARS-CoV-2 drug screening methods, our system is more economical and convenient, facilitating the development of more potent and selective nsp13 inhibitors and enabling the discovery of new antiviral therapies.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Nucleosídeo-Trifosfatase/genética , RNA Helicases/metabolismo , Proteínas não Estruturais Virais/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Antivirais/farmacologia , DNA , Plasmídeos/genéticaRESUMO
Plasmid families harbor different maintenances functions, depending on their size and copy number. Low copy number plasmids rely on active partition systems, organizing a partition complex at specific centromere sites that is actively positioned using NTPase proteins. Some low copy number plasmids lack an active partition system, but carry atypical intracellular positioning systems using a single protein that binds to the centromere site but without an associated NTPase. These systems have been studied in the case of the Escherichia coli R388 and of the Staphylococcus aureus pSK1 plasmids. Here we review these two systems, which appear to be unrelated but share common features, such as their distribution on plasmids of medium size and copy number, certain activities of their centromere-binding proteins, StbA and Par, respectively, as well as their mode of action, which may involve dynamic interactions with the nucleoid-packed chromosome of their hosts.
Assuntos
Variações do Número de Cópias de DNA , Nucleosídeo-Trifosfatase , Humanos , Plasmídeos/genética , Nucleosídeo-Trifosfatase/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Bactérias/genética , Segregação de CromossomosRESUMO
The CRESS-DNA viruses are the ubiquitous virus detected in almost all eukaryotic life trees and play an essential role in the maintaining ecosystem of the globe. Still, their genetic diversity is not fully understood. Here, we bring to light the genetic diversity of replication (Rep) and capsid (Cap) proteins of CRESS-DNA viruses. We divided the Rep protein of the CRESS-DNA virus into 10 clusters using CLANS and phylogenetic analyses. Also, most of the Rep protein in Rep cluster 1 (R1) and R2 (Circoviridae, Smacoviridae, Nanoviridae, and CRESSV1-5) contain the Viral_Rep superfamily and P-loop_NTPase superfamily domains, while the Rep protein of viruses in other clusters has no such characterized functional domain. The Circoviridae, Nanoviridae, and CRESSV1-3 viruses contain two domains, such as Viral_Rep and P-loop_NTPase; the CRESSV4 and CRESSV5 viruses have only the Viral_Rep domain; most of the sequences in the pCRESS-related group have only P-loop_NTPase; and Smacoviridae do not have these two domains. Further, we divided the Cap protein of the CRESS-DNA virus into 20 clusters using CLANS and phylogenetic analyses. The Rep and Cap proteins of Circoviridae and Smacoviridae are grouped into a specific cluster. Cap protein of CRESS-DNA viruses grouped with one cluster and Rep protein with another cluster. Further, our study reveals that selection pressure plays a significant role in the evolution of CRESS-DNA viruses' Rep and Cap genes rather than mutational pressure. We hope this study will help determine the genetic diversity of CRESS-DNA viruses as more sequences are discovered in the future. IMPORTANCE The genetic diversity of CRESS-DNA viruses is not fully understood. CRESS-DNA viruses are classified as CRESSV1 to CRESSV6 using only Rep protein. This study revealed that the Rep protein of the CRESS-DNA viruses is classified as CRESSV1 to CRESSV6 groups and the new Smacoviridae-related, CRESSV2-related, pCRESS-related, Circoviridae-related, and 1 to 4 outgroups, according to the Viral_Rep and P-loop_NTPase domain organization, CLANS, and phylogenetic analysis. Furthermore, for the first time in this study, the Cap protein of CRESS-DNA viruses was classified into 20 distinct clusters by CLANS and phylogenetic analysis. Through this classification, the genetic diversity of CRESS-DNA viruses clarifies the possibility of recombinations in Cap and Rep proteins. Finally, it has been shown that selection pressure plays a significant role in the evolution and genetic diversity of Cap and Rep proteins. This study explains the genetic diversity of CRESS-DNA viruses and hopes that it will help classify future detected viruses.
Assuntos
Brassicaceae , DNA Viral , DNA Viral/genética , Filogenia , Brassicaceae/genética , Ecossistema , Nucleosídeo-Trifosfatase/genética , Vírus de DNA/genética , Proteínas do Capsídeo/genética , Variação Genética , Genoma Viral , DNA CircularRESUMO
RNA-remodeling proteins, including RNA helicases and chaperones, play vital roles in the remodeling of structured RNAs. During viral replication, viruses require RNA-remodeling proteins to facilitate proper folding and/or re-folding the viral RNA elements. Coxsackieviruses B3 (CVB3) and Coxsackieviruses B5 (CVB5), belonging to the genus Enterovirus in the family Picornaviridae, have been reported to cause various infectious diseases such as hand-foot-and-mouth disease, aseptic meningitis, and viral myocarditis. However, little is known about whether CVB3 and CVB5 encode any RNA remodeling proteins. In this study, we showed that 2C proteins of CVB3 and CVB5 contained the conserved SF3 helicase A, B, and C motifs, and functioned not only as RNA helicase that unwound RNA helix bidirectionally in an NTP-dependent manner, but also as RNA chaperone that remodeled structured RNAs and facilitated RNA strand annealing independently of NTP. In addition, we determined that the NTPase activity and RNA helicase activity of 2C proteins of CVB3 and CVB5 were dependent on the presence of divalent metallic ions. Our findings demonstrate that 2C proteins of CVBs possess RNA-remodeling activity and underline the functional importance of 2C protein in the life cycle of CVBs.
Assuntos
Enterovirus Humano B , RNA Helicases , Animais , Enterovirus Humano B/genética , Nucleosídeo-Trifosfatase/genética , RNA Helicases/genética , RNA Helicases/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Replicação ViralRESUMO
The duck hepatitis A virus 1 (DHAV-1) 2C protein was predicted to be a superfamily III helicase member and includes nucleotide binding (NTB) and putative RNA helicase activity motifs. To study whether DHAV-1 2C protein has NTB activity, we expressed DHAV-1 2C protein with maltose binding protein (MBP) to solve its poor solubility in a prokaryotic expression system. We showed that the DHAV-1 2C protein has nucleoside triphosphatase (NTPase) activity by measuring the released phosphate. The NTPase of the DHAV-1 2C protein is Mg2+ indispensable and affected by other biochemical characteristics such as Mn2+, Ca2+, Zn2+, Na+ and pH. Guanidine hydrochloride (GdnHCl), a potent inhibitor of viral RNA replication, inhibited ATPase activity of the DHAV-1 2C protein in a dose-dependent manner. Finally, we constructed three mutants to identify the key site for the ATPase activity of the DHAV-1 2C protein. These results indicate that lysine at position 151 of the DHAV-1 2C protein is very important for NTPase activity. Here, we demonstrated and partially characterized that the DHAV-1 2C protein has NTPase activity and showed that mutation of the lysine in the conserved Walker A impairs that activity. The results serve to confirm what is readily predicted from previous work on picornavirus 2C proteins. It also provides a basis for further study of the 2C protein and the function of NTPase activity on the viral life cycle.
Assuntos
Proteínas de Transporte , Vírus da Hepatite do Pato , Lisina , Nucleosídeo-Trifosfatase , Proteínas não Estruturais Virais , Animais , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Patos , Vírus da Hepatite do Pato/genética , Lisina/metabolismo , Nucleosídeo-Trifosfatase/genética , Nucleosídeo-Trifosfatase/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/genéticaRESUMO
The human norovirus (HuNV)-encoded nucleoside-triphosphatase (NTPase) is a multifunctional protein critically involved in viral replication and pathogenesis. Previously, we have shown that the viral NTPase is capable of forming vesicle clusters in cells, interacting with other viral proteins such as P22, and promoting cellular apoptosis. Herein, we demonstrate that NTPase-associated vesicle clusters correspond to lipid droplets (LDs) wrapped by the viral protein and show that NTPase-induced apoptosis is mediated through both caspase-8- and caspase-9-dependent pathways. Deletion analysis revealed that the N-terminal 179-amino-acid (aa) region of NTPase encompasses two LD-targeting motifs (designated LTM-1 and LTM-2), two apoptosis-inducing motifs, and multiple regulatory regions. Interestingly, the identified LTM-1 and LTM-2, which are located from aa 1 to 50 and from aa 51 to 90, respectively, overlap with the two apoptosis-inducing motifs. Although there was no positive correlation between the extent of LD localization and the degree of cellular apoptosis for NTPase mutants, we noticed that mutant proteins defective in LD-targeting ability could not induce cellular apoptosis. In addition to LD targeting, the amphipathic LTM-1 and LTM-2 motifs could have the potential to direct fusion proteins to the endoplasmic reticulum (ER). Furthermore, we found that the LTM-1 motif is a P22-interacting motif. However, P22 functionally augmented the proapoptotic activity of the LTM-2 fusion protein but not the LTM-1 fusion protein. Overall, our findings propose that NTPase may participate in multiple cellular processes through binding to LDs or to the ER via its N-terminal amphipathic helix motifs. IMPORTANCE Human noroviruses (HuNVs) are the major agent of global gastroenteritis outbreaks. However, due to the lack of an efficient cell culture system for HuNV propagation, functions of the viral-encoded proteins in host cells are still poorly understood. In the current study, we present that the viral NTPase is a lipid droplet (LD)-associated protein, and we identify two LD-targeting motifs, LTM-1 and LTM-2, in its N-terminal domain. In particular, the identified LTM-1 and LTM-2 motifs, which contain a hydrophobic region and an amphipathic helix, are also capable of delivering the fusion protein to the endoplasmic reticulum (ER), promoting cellular apoptosis, and physically or functionally associating with another viral protein P22. Since LDs and the ER have been linked to several biological functions in cells, our study therefore proposes that the norovirus NTPase may utilize LDs or the ER as replication platforms to benefit viral replication and pathogenesis.
Assuntos
Gotículas Lipídicas/metabolismo , Norovirus/enzimologia , Nucleosídeo-Trifosfatase/isolamento & purificação , Proteínas Virais/metabolismo , Apoptose , Retículo Endoplasmático/metabolismo , Gastroenterite , Humanos , Norovirus/genética , Nucleosídeo-Trifosfatase/genética , Replicação ViralRESUMO
IM30, the inner membrane-associated protein of 30 kDa, is conserved in cyanobacteria and chloroplasts. Although its exact physiological function is still mysterious, IM30 is clearly essential for thylakoid membrane biogenesis and/or dynamics. Recently, a cryptic IM30 GTPase activity has been reported, albeit thus far no physiological function has been attributed to this. Yet, it is still possible that GTP binding/hydrolysis affects formation of the prototypical large homo-oligomeric IM30 ring and rod structures. Here, we show that the Synechocystis sp. PCC 6803 IM30 protein in fact is an NTPase that hydrolyzes GTP and ATP, but not CTP or UTP, with about identical rates. While IM30 forms large oligomeric ring complexes, nucleotide binding and/or hydrolysis are clearly not required for ring formation.
Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas de Membrana/metabolismo , Nucleosídeo-Trifosfatase/metabolismo , Synechocystis/enzimologia , Tilacoides/enzimologia , Trifosfato de Adenosina/química , Proteínas de Bactérias/genética , Clonagem Molecular , Ensaios Enzimáticos , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Guanosina Trifosfato/química , Hidrólise , Cinética , Proteínas de Membrana/genética , Microscopia Eletrônica , Nucleosídeo-Trifosfatase/genética , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Synechocystis/genética , Synechocystis/ultraestrutura , Tilacoides/genética , Tilacoides/ultraestruturaRESUMO
During their maturation, nascent 40S subunits enter a translation-like quality control cycle, where they are joined by mature 60S subunits to form 80S-like ribosomes. While these assembly intermediates are essential for maturation and quality control, how they form, and how their structure promotes quality control, remains unknown. To address these questions, we determined the structure of an 80S-like ribosome assembly intermediate to an overall resolution of 3.4 Å. The structure, validated by biochemical data, resolves a large body of previously paradoxical data and illustrates how assembly and translation factors cooperate to promote the formation of an interface that lacks many mature subunit contacts but is stabilized by the universally conserved methyltransferase Dim1. We also show how Tsr1 enables this interface by blocking the canonical binding of eIF5B to 40S subunits, while maintaining its binding to 60S. The structure also shows how this interface leads to unfolding of the platform, which allows for temporal regulation of the ATPase Fap7, thus linking 40S maturation to quality control during ribosome assembly.
Assuntos
Adenilato Quinase/genética , Regulação Fúngica da Expressão Gênica , Metiltransferases/genética , Proteínas Nucleares/genética , Nucleosídeo-Trifosfatase/genética , Proteínas Ribossômicas/genética , Subunidades Ribossômicas Maiores de Eucariotos/genética , Subunidades Ribossômicas Menores de Eucariotos/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Adenilato Quinase/química , Adenilato Quinase/metabolismo , Sítios de Ligação , Metiltransferases/química , Metiltransferases/metabolismo , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Nucleosídeo-Trifosfatase/química , Nucleosídeo-Trifosfatase/metabolismo , Biogênese de Organelas , Ligação Proteica , Biossíntese de Proteínas , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/ultraestrutura , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/ultraestrutura , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Among Flaviviridae, in West Nile virus (WNV) and Hepatitis C virus (HCV), the non-structural protein NS4A modulates the NTPase activity of viral helicases during nucleic acid unwinding through its N-terminal disordered residues (1-50). In HCV, the acidic NS4A also serves as a cofactor for regulating the NS3 protease activity. However, in case of Zika virus (ZIKV), the role of NS4A and its impact on activities of NS3 helicase and protease is not known. In order to elucidate the role of NS4A, we checked the NTPase activity of NS3 helicase and protease activity of NS3 protease in presence of NS4A N-terminal region (residues 1-48) peptide. Our enzyme kinetics results together with binding experiment clearly demonstrate that NS3 helicase in presence of NS4A peptide increased the rate of ATP hydrolysis whereas the protease activity of NS3 protease was not affected. Therefore, like WNV and HCV, our results establish a role of ZIKV NS4A being a cofactor for modulating the NTPase activity of ZIKV NS3 helicase.
Assuntos
Nucleosídeo-Trifosfatase/química , RNA Helicases/química , Serina Endopeptidases/química , Proteínas Virais/química , Zika virus/enzimologia , Coenzimas , Nucleosídeo-Trifosfatase/genética , Domínios Proteicos , RNA Helicases/genética , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Zika virus/genéticaRESUMO
NOD-like receptors (NLRs) are traditionally recognized as major inflammasome components. The role of NLRs in germ cell differentiation and reproduction is not known. Here, we identified the gonad-specific Nlrp14 as a pivotal regulator in primordial germ cell-like cell (PGCLC) differentiation in vitro. Physiologically, knock out of Nlrp14 resulted in reproductive failure in both female and male mice. In adult male mice, Nlrp14 knockout (KO) inhibited differentiation of spermatogonial stem cells (SSCs) and meiosis, resulting in trapped SSCs in early stages, severe oligozoospermia, and sperm abnormality. Mechanistically, NLRP14 promoted spermatogenesis by recruiting a chaperone cofactor, BAG2, to bind with HSPA2 and form the NLRP14-HSPA2-BAG2 complex, which strongly inhibited ChIP-mediated HSPA2 polyubiquitination and promoted its nuclear translocation. Finally, loss of HSPA2 protection and BAG2 recruitment by NLRP14 was confirmed in a human nonsense germline variant associated with male sterility. Together, our data highlight a unique proteasome-mediated, noncanonical function of NLRP14 in PGCLC differentiation and spermatogenesis, providing mechanistic insights of gonad-specific NLRs in mammalian germline development.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Diferenciação Celular/fisiologia , Proteínas de Choque Térmico HSP70/metabolismo , Chaperonas Moleculares/metabolismo , Espermatogênese/genética , Transporte Ativo do Núcleo Celular/genética , Transporte Ativo do Núcleo Celular/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Células-Tronco Germinativas Adultas/fisiologia , Animais , Proteínas Reguladoras de Apoptose/genética , Feminino , Deleção de Genes , Regulação da Expressão Gênica/fisiologia , Variação Genética , Células Germinativas , Proteínas de Choque Térmico HSP70/genética , Humanos , Infertilidade Masculina/genética , Masculino , Camundongos , Chaperonas Moleculares/genética , Nucleosídeo-Trifosfatase/genética , Nucleosídeo-Trifosfatase/metabolismo , Espermatogênese/fisiologiaRESUMO
African swine fever virus (ASFV) is the causative agent of the African swine fever (ASF) epizootic currently affecting pigs throughout Eurasia, causing significant economic losses in the swine industry. The virus genome encodes for more than 160 genes, of which only a few have been studied in detail. Here we describe the previously uncharacterized ASFV open reading frame (ORF) C962R, a gene encoding for a putative NTPase. RNA transcription studies using infected swine macrophages demonstrate that the C962R gene is translated as a late virus protein. A recombinant ASFV lacking the C962R gene (ASFV-G-ΔC962R) demonstrates in vivo that the C962R gene is non-essential, since ASFV-G-ΔC962R has similar replication kinetics in primary swine macrophage cell cultures when compared to parental highly virulent field isolate Georgia2007 (ASFV-G). Experimental infection of domestic pigs with ASFV-G-ΔC962R produced a clinical disease similar to that caused by the parental ASFV-G, confirming that deletion of the C962R gene from the ASFV genome does not impact virulence.
Assuntos
Vírus da Febre Suína Africana/genética , Vírus da Febre Suína Africana/patogenicidade , Febre Suína Africana/patologia , Nucleosídeo-Trifosfatase/genética , Sequência de Aminoácidos , Animais , Células Cultivadas , Deleção de Genes , Genoma Viral/genética , Macrófagos/virologia , Fases de Leitura Aberta/genética , Alinhamento de Sequência , Suínos , Doenças dos Suínos/virologia , Proteínas Virais/genética , Fatores de Virulência/genética , Replicação Viral/genéticaRESUMO
The ongoing outbreak of Coronavirus Disease 2019 (COVID-19) has become a global public health emergency. SARS-coronavirus-2 (SARS-CoV-2), the causative pathogen of COVID-19, is a positive-sense single-stranded RNA virus belonging to the family Coronaviridae. For RNA viruses, virus-encoded RNA helicases have long been recognized to play pivotal roles during viral life cycles by facilitating the correct folding and replication of viral RNAs. Here, our studies show that SARS-CoV-2-encoded nonstructural protein 13 (nsp13) possesses the nucleoside triphosphate hydrolase (NTPase) and RNA helicase activities that can hydrolyze all types of NTPs and unwind RNA helices dependently of the presence of NTP, and further characterize the biochemical characteristics of these two enzymatic activities associated with SARS-CoV-2 nsp13. Moreover, we found that some bismuth salts could effectively inhibit both the NTPase and RNA helicase activities of SARS-CoV-2 nsp13 in a dose-dependent manner. Thus, our findings demonstrate the NTPase and helicase activities of SARS-CoV-2 nsp13, which may play an important role in SARS-CoV-2 replication and serve as a target for antivirals.
Assuntos
Betacoronavirus/metabolismo , Bismuto/farmacologia , Metiltransferases/metabolismo , Nucleosídeo-Trifosfatase/efeitos dos fármacos , RNA Helicases/efeitos dos fármacos , Sais/farmacologia , Proteínas não Estruturais Virais/metabolismo , Adenosina Trifosfatases/efeitos dos fármacos , Adenosina Trifosfatases/metabolismo , Betacoronavirus/enzimologia , Betacoronavirus/genética , COVID-19 , Infecções por Coronavirus/virologia , Humanos , Metiltransferases/genética , Nucleosídeo-Trifosfatase/genética , Nucleosídeo-Trifosfatase/metabolismo , Pandemias , Pneumonia Viral/virologia , RNA Helicases/genética , RNA Helicases/metabolismo , Proteínas Recombinantes , SARS-CoV-2 , Síndrome Respiratória Aguda Grave , Proteínas não Estruturais Virais/genética , Replicação ViralRESUMO
N6-Methyladenosine (m6A) RNA methylation plays important roles during development in different species. However, knowledge of m6A RNA methylation in monocots remains limited. In this study, we reported that OsFIP and OsMTA2 are the components of m6A RNA methyltransferase complex in rice and uncovered a previously unknown function of m6A RNA methylation in regulation of plant sporogenesis. Importantly, OsFIP is essential for rice male gametogenesis. Knocking out of OsFIP results in early degeneration of microspores at the vacuolated pollen stage and simultaneously causes abnormal meiosis in prophase I. We further analyzed the profile of rice m6A modification during sporogenesis in both WT and OsFIP loss-of-function plants, and identified a rice panicle specific m6A modification motif "UGWAMH". Interestingly, we found that OsFIP directly mediates the m6A methylation of a set of threonine protease and NTPase mRNAs and is essential for their expression and/or splicing, which in turn regulates the progress of sporogenesis. Our findings revealed for the first time that OsFIP plays an indispensable role in plant early sporogenesis. This study also provides evidence for the different functions of the m6A RNA methyltransferase complex between rice and Arabidopsis.
Assuntos
Gametogênese Vegetal , Regulação da Expressão Gênica de Plantas , Metiltransferases/genética , Oryza/genética , Proteínas de Plantas/genética , Subunidades Proteicas/genética , Adenosina/análogos & derivados , Motivos de Aminoácidos , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Mutação com Perda de Função , Prófase Meiótica I , Metilação , Metiltransferases/metabolismo , Nucleosídeo-Trifosfatase/genética , Nucleosídeo-Trifosfatase/metabolismo , Oryza/crescimento & desenvolvimento , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Pólen/genética , Pólen/crescimento & desenvolvimento , Pólen/metabolismo , Subunidades Proteicas/metabolismo , RNA de Plantas , Especificidade da EspécieRESUMO
The ABC transporter Pdr5 of S. cerevisiae is a key player of the PDR network that works as a first line of defense against a wide range of xenobiotic compounds. As the first discovered member of the family of asymmetric PDR ABC transporters, extensive studies have been carried out to elucidate the molecular mechanism of drug efflux and the details of the catalytic cycle. Pdr5 turned out to be an excellent model system to study functional and structural characteristics of asymmetric, uncoupled ABC transporters. However, to date studies have been limited to in vivo or plasma membrane systems, as it was not possible to isolate Pdr5 in a functional state. Here, we describe the solubilization and purification of Pdr5 to homogeneity in a functional state as confirmed by in vitro assays. The ATPase deficient Pdr5 E1036Q mutant was used as a control and proves that detergent-purified wild-type Pdr5 is functional resembling in its activity the one in its physiological environment. Finally, we show that the isolated active Pdr5 is monomeric in solution. Taken together, our results described in this study will enable a variety of functional investigations on Pdr5 required to determine molecular mechanism of this asymmetric ABC transporter.
Assuntos
Transportadores de Cassetes de Ligação de ATP/isolamento & purificação , Transportadores de Cassetes de Ligação de ATP/metabolismo , Nucleosídeo-Trifosfatase/metabolismo , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Nucleosídeo-Trifosfatase/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismoRESUMO
Ebola virus (EBOV) is a non-segmented, negative-sense RNA virus (NNSV) in the family Filoviridae, and is recognized as one of the most lethal pathogens in the planet. For RNA viruses, cellular or virus-encoded RNA helicases play pivotal roles in viral life cycles by remodelling viral RNA structures and/or unwinding viral dsRNA produced during replication. However, no helicase or helicase-like activity has ever been found to associate with any NNSV-encoded proteins, and it is unknown whether the replication of NNSVs requires the participation of any viral or cellular helicase. Here, we show that despite of containing no conserved NTPase/helicase motifs, EBOV VP35 possesses the NTPase and helicase-like activities that can hydrolyse all types of NTPs and unwind RNA helices in an NTP-dependent manner, respectively. Moreover, guanidine hydrochloride, an FDA-approved compound and inhibitor of certain viral helicases, inhibited the NTPase and helicase-like activities of VP35 as well as the replication/transcription of an EBOV minigenome replicon in cells, highlighting the importance of VP35 helicase-like activity during EBOV life cycle. Together, our findings provide the first demonstration of the NTPase/helicase-like activity encoded by EBOV, and would foster our understanding of EBOV and NNSVs.
Assuntos
Ebolavirus/genética , Ebolavirus/metabolismo , Doença pelo Vírus Ebola/virologia , Nucleoproteínas/fisiologia , RNA de Cadeia Dupla/química , Proteínas do Core Viral/fisiologia , Trifosfato de Adenosina/química , Motivos de Aminoácidos , Células Cultivadas , DNA Helicases/metabolismo , Humanos , Proteínas do Nucleocapsídeo , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , Nucleosídeo-Trifosfatase/genética , Ligação Proteica , RNA Helicases/metabolismo , RNA Viral/metabolismo , Proteínas do Core Viral/genética , Proteínas do Core Viral/metabolismo , Proteínas não Estruturais Virais/metabolismo , Replicação ViralRESUMO
BACKGROUND: Cellular metabolism generates reactive oxygen species. The oxidation and deamination of the deoxynucleoside triphosphate (dNTP) pool results in the formation of non-canonical, toxic dNTPs that can cause mutations, genome instability, and cell death. House-cleaning or sanitation enzymes that break down and detoxify non-canonical nucleotides play major protective roles in nucleotide metabolism and constitute key drug targets for cancer and various pathogens. We hypothesized that owing to their protective roles in nucleotide metabolism, these house-cleaning enzymes are key drug targets in the malaria parasite. METHODS: Using the rodent malaria parasite Plasmodium berghei we evaluate here, by gene targeting, a group of conserved proteins with a putative function in the detoxification of non-canonical nucleotides as potential antimalarial drug targets: they are inosine triphosphate pyrophosphatase (ITPase), deoxyuridine triphosphate pyrophosphatase (dUTPase) and two NuDiX hydroxylases, the diadenosine tetraphosphate (Ap4A) hydrolase and the nucleoside triphosphate hydrolase (NDH). RESULTS: While all four proteins are expressed constitutively across the intraerythrocytic developmental cycle, neither ITPase nor NDH are required for parasite viability. dutpase and ap4ah null mutants, on the other hand, are not viable suggesting an essential function for these proteins for the malaria parasite. CONCLUSIONS: Plasmodium dUTPase and Ap4A could be drug targets in the malaria parasite.
Assuntos
Hidrolases Anidrido Ácido/genética , Malária/parasitologia , Plasmodium berghei/enzimologia , Pirofosfatases/genética , Hidrolases Anidrido Ácido/metabolismo , Animais , Antimaláricos/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Nucleosídeo-Trifosfatase/genética , Nucleosídeo-Trifosfatase/metabolismo , Plasmodium berghei/genética , Pirofosfatases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Inosina TrifosfataseRESUMO
Eukaryotic DExH-box proteins are important post-transcriptional gene regulators, many of which employ RNA-stimulated nucleoside triphosphatase activity to remodel RNAs or ribonucleoprotein complexes. However, bacterial DExH-box proteins are structurally and functionally poorly characterized. We report the crystal structure of the Escherichia coli DExH-box protein HrpB. A globular head is composed of dual RecA, winged-helix, helical bundle and oligonucleotide/oligosaccharide-binding domains, resembling a compact version of eukaryotic DExH-box proteins. Additionally, HrpB harbors a C-terminal region not found in proteins with known structure, which bestows the protein with unique interaction potential. Interaction and activity assays showed that the protein binds RNA but not DNA, hydrolyzes all nucleoside triphosphates in an RNA-stimulated manner, but does not unwind diverse model RNAs in vitro. These observations can be rationalized by detailed comparisons with structurally characterized eukaryotic DExH-box proteins. Comparative phenotypic analyses of an E. coli hrpB knockout mutant suggested diverse functions of HrpB homologs in different bacteria.
Assuntos
RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Nucleosídeo-Trifosfatase/química , Nucleosídeo-Trifosfatase/metabolismo , Oligossacarídeos/metabolismo , RNA Bacteriano/metabolismo , Sítios de Ligação , Cristalografia por Raios X , RNA Helicases DEAD-box/genética , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Modelos Moleculares , Nucleosídeo-Trifosfatase/genética , Ligação Proteica , Domínios Proteicos , Estrutura Secundária de Proteína , Especificidade por SubstratoRESUMO
Nucleoside triphosphate diphosphohydrolases (NTPDases) are enzymes that belong to the GDA1/CD39 protein superfamily. These enzymes catalyze the hydrolysis of ATP and ADP to the monophosphate form (AMP). Biochemical characterization of the nucleotidases/NTPDases from various types of cells, including those from plants, animals, and pathogenic organisms, has revealed the existence of several isoforms with different specificities with respect to divalent cations (magnesium, calcium, manganese, and zinc) and substrates. In mammals, the NTPDases play important roles in the regulation of thrombosis and inflammation. In parasites of the genus Leishmania, the causative agents of leishmaniasis, two NTPDase isoforms, termed NTPDase-1 and NTPDase-2 have been described. Independently of their cellular localization, whether cell-surface localized, secreted or targeted to other organelles, in some Leishmania species these NTPDases could be involved in parasite growth, infectivity, and virulence. Experimental evidence has suggested that the hydrolysis of ATP and ADP by parasite ecto-nucleotidases can down-modulate the host immune response. In this context, the present work provides an overview of recent works that show strong evidence not only of the involvement of the nucleotidases/NTPDases in Leishmania spp infectivity and virulence but also of the molecular mechanisms that lead to the success of the parasitic infection.
Assuntos
Leishmania/enzimologia , Nucleosídeo-Trifosfatase/metabolismo , Proteínas de Protozoários/metabolismo , Animais , Antígenos CD/química , Antígenos CD/metabolismo , Apirase/química , Apirase/metabolismo , Humanos , Leishmania/imunologia , Leishmania/fisiologia , Leishmaniose/parasitologia , Leishmaniose/patologia , Leishmaniose/veterinária , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/metabolismo , Nucleosídeo-Trifosfatase/química , Nucleosídeo-Trifosfatase/genética , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , VirulênciaRESUMO
The genotype II.4 (GII.4) variants of human noroviruses (HuNVs) are recognized as the major agent of global gastroenteritis outbreaks. Due to the lack of an efficient cell culture system for HuNV propagation, the exact roles of HuNV-encoded nonstructural proteins (including Nterm, NTPase, P22, VPg, Pro, and RdRp) in viral replication or pathogenesis have not yet been fully understood. Here, we report the molecular characterization of the GII.4 HuNV-encoded NTPase (designated GII-NTPase). Results from our studies showed that GII-NTPase forms vesicular or nonvesicular textures in the cell cytoplasm, and the nonvesicular fraction of GII-NTPase significantly localizes to the endoplasmic reticulum (ER) or mitochondria. Deletion analysis revealed that the N-terminal 179-amino-acid (aa) region of GII-NTPase is required for vesicle formation and for ER colocalization, whereas the C-terminal region is involved in mitochondrial colocalization. In particular, two mitochondrion-targeting domains were identified in the C-terminal region of GII-NTPase which perfectly colocalized with mitochondria when the N-terminal region of GII-NTPase was deleted. However, the corresponding C-terminal portions of NTPase derived from the GI HuNV did not show mitochondrial colocalization. We also found that GII-NTPase physically interacts with itself as well as with Nterm and P22, but not VPg, Pro, and RdRp, in cells. The Nterm- and P22-interacting region was mapped to the N-terminal 179-aa region of GII-NTPase, whereas the self-assembly of GII-NTPase could be achieved via a head-to-head, tail-to-tail, or head-to-tail configuration. More importantly, we demonstrate that GII-NTPase possesses a proapoptotic activity, which can be further enhanced by coexpression with Nterm or P22.IMPORTANCE Despite the importance of human norovirus GII.4 variants in global gastroenteritis outbreaks, the basic biological functions of the viral nonstructural proteins in cells remain rarely investigated. In this report, we focus our studies on characteristics of the GII.4 norovirus-encoded NTPase (GII-NTPase). We unexpectedly find that GII-NTPase can perfectly colocalize with mitochondria after its N-terminal region is deleted. However, such a phenomenon is not observed for NTPase encoded by a GI strain. We further reveal that the N-terminal 179-aa region of GII-NTPase is sufficient to mediate (i) vesicle formation, (ii) ER colocalization, (iii) the interaction with two other nonstructural proteins, including Nterm and P22, (iv) the formation of homodimers or homo-oligomers, and (v) the induction of cell apoptosis. Taken together, our findings emphasize that the virus-encoded NTPase must have multiple activities during viral replication or pathogenesis; however, these activities may vary somewhat among different genogroups.