RESUMEN
RNA-dependent RNA polymerase 3Dpol is a key enzyme for the replication of picornaviruses. The viral genome is translated into a single polyprotein that is subsequently proteolytically processed into matured products. The 3Dpol enzyme arises from a stable 3CD precursor that has high proteolytic activity but no polymerase activity. Upon cleavage of the precursor the newly established N-terminus of 3Dpol is liberated and inserts itself into a pocket on the surface of the 3Dpol enzyme. The essential residue for this mechanism is the very first glycine that is conserved among almost all picornaviruses. However, kobuviruses and siciniviruses have a serine residue instead. Intrigued by this anomaly we sought to solve the crystal structure of these 3Dpol enzymes. The structures revealed a unique fold of the 3Dpol N-termini but the very first serine residues were inserted into a charged pocket in a similar manner as the glycine residue in other picornaviruses. These structures revealed a common underlying mechanism of 3Dpol activation that lies in activation of the α10 helix containing a key catalytical residue Asp238 that forms a hydrogen bond with the 2' hydroxyl group of the incoming NTP nucleotide.
Asunto(s)
Kobuvirus/enzimología , Picornaviridae/enzimología , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas Virales/metabolismo , Cristalografía por Rayos X , Citometría de Flujo , Células HeLa , Humanos , Enlace de Hidrógeno , Mutagénesis Sitio-Dirigida , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/genética , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
Zika virus is a global health threat due to significantly elevated risk of fetus malformations in infected pregnant women. Currently, neither an effective therapy nor a prophylactic vaccination is available for clinical use, desperately necessitating novel therapeutics and approaches to obtain them. Here, we present a structural model of the Zika virus RNA-dependent RNA polymerase (ZIKV RdRp) in complex with template and nascent RNAs, Mg2+ ions and accessing nucleoside triphosphate. The model allowed for docking studies aimed at effective pre-screening of potential inhibitors of ZIKV RdRp. Applicability of the structural model for docking studies was illustrated with the NITD008 artificial nucleotide that is known to effectively inhibit the function of the ZIKV RdRp. The ZIKV RdRp - RNA structural model is provided for all possible variations of the nascent RNA bases pairs to enhance its general utility in docking and modelling experiments. The developed model makes the rational design of novel nucleosides and nucleotide analogues feasible and thus provides a solid platform for the development of advanced antiviral therapy.
Asunto(s)
ARN Polimerasa Dependiente del ARN/química , ARN/química , Infección por el Virus Zika/genética , Virus Zika/química , Adenosina/análogos & derivados , Adenosina/química , Adenosina/farmacología , Humanos , Magnesio/química , Modelos Moleculares , Simulación del Acoplamiento Molecular , Nucleósidos/química , Nucleótidos/química , Polifosfatos/química , Conformación Proteica/efectos de los fármacos , ARN/genética , ARN Polimerasa Dependiente del ARN/genética , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genética , Replicación Viral/genética , Virus Zika/genética , Virus Zika/patogenicidad , Infección por el Virus Zika/virologíaRESUMEN
Most single stranded plus RNA viruses hijack phosphatidylinositol 4-kinases (PI4Ks) to generate membranes highly enriched in phosphatidylinositol 4-phosphate (PI4P). These membranous compartments known as webs, replication factories or replication organelles are essential for viral replication because they provide protection from the innate intracellular immune response while serving as platforms for viral replication. Using purified recombinant proteins and biomimetic model membranes we show that the nonstructural viral 3A protein is sufficient to promote membrane hyper-phosphorylation given the proper intracellular cofactors (PI4KB and ACBD3). However, our bio-mimetic in vitro reconstitution assay revealed that rather than the presence of PI4P specifically, negative charge alone is sufficient for the recruitment of 3Dpol enzymes to the surface of the lipid bilayer. Additionally, we show that membrane tethered viral 3B protein (also known as Vpg) works in combination with the negative charge to increase the efficiency of membrane recruitment of 3Dpol.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Membrana Celular/metabolismo , Kobuvirus/enzimología , Proteínas de la Membrana/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Infecciones por Picornaviridae/metabolismo , Proteínas no Estructurales Virales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Humanos , Proteínas de la Membrana/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Infecciones por Picornaviridae/virología , Proteínas no Estructurales Virales/genéticaRESUMEN
Phosphatidylinositol 4-kinase beta (PI4KB) is one of four human PI4K enzymes that generate phosphatidylinositol 4-phosphate (PI4P), a minor but essential regulatory lipid found in all eukaryotic cells. To convert their lipid substrates, PI4Ks must be recruited to the correct membrane compartment. PI4KB is critical for the maintenance of the Golgi and trans Golgi network (TGN) PI4P pools, however, the actual targeting mechanism of PI4KB to the Golgi and TGN membranes is unknown. Here, we present an NMR structure of the complex of PI4KB and its interacting partner, Golgi adaptor protein acyl-coenzyme A binding domain containing protein 3 (ACBD3). We show that ACBD3 is capable of recruiting PI4KB to membranes both in vitro and in vivo, and that membrane recruitment of PI4KB by ACBD3 increases its enzymatic activity and that the ACBD3:PI4KB complex formation is essential for proper function of the Golgi.