RESUMO
Rift Valley fever virus (RVFV), a Phlebovirus with a genome consisting of three single-stranded RNA segments, is spread by infected mosquitoes and causes large viral outbreaks in Africa. RVFV encodes a nucleoprotein (N) that encapsidates the viral RNA. The N protein is the major component of the ribonucleoprotein complex and is also required for genomic RNA replication and transcription by the viral polymerase. Here we present the 1.6 Å crystal structure of the RVFV N protein in hexameric form. The ring-shaped hexamers form a functional RNA binding site, as assessed by mutagenesis experiments. Electron microscopy (EM) demonstrates that N in complex with RNA also forms rings in solution, and a single-particle EM reconstruction of a hexameric N-RNA complex is consistent with the crystallographic N hexamers. The ring-like organization of the hexamers in the crystal is stabilized by circular interactions of the N terminus of RVFV N, which forms an extended arm that binds to a hydrophobic pocket in the core domain of an adjacent subunit. The conformation of the N-terminal arm differs from that seen in a previous crystal structure of RVFV, in which it was bound to the hydrophobic pocket in its own core domain. The switch from an intra- to an inter-molecular interaction mode of the N-terminal arm may be a general principle that underlies multimerization and RNA encapsidation by N proteins from Bunyaviridae. Furthermore, slight structural adjustments of the N-terminal arm would allow RVFV N to form smaller or larger ring-shaped oligomers and potentially even a multimer with a super-helical subunit arrangement. Thus, the interaction mode between subunits seen in the crystal structure would allow the formation of filamentous ribonucleocapsids in vivo. Both the RNA binding cleft and the multimerization site of the N protein are promising targets for the development of antiviral drugs.
Assuntos
Proteínas do Nucleocapsídeo/química , Multimerização Proteica , RNA Viral/química , Ribonucleoproteínas/química , Vírus da Febre do Vale do Rift/fisiologia , Sequência de Aminoácidos , Animais , Cristalografia por Raios X/métodos , DNA Complementar/genética , Humanos , Microscopia Eletrônica , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas do Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/isolamento & purificação , Proteínas do Nucleocapsídeo/ultraestrutura , Domínios e Motivos de Interação entre Proteínas , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/ultraestrutura , Vírus da Febre do Vale do Rift/química , Vírus da Febre do Vale do Rift/genética , Vírus da Febre do Vale do Rift/ultraestrutura , Alinhamento de Sequência , Ressonância de Plasmônio de Superfície/métodos , Montagem de VírusRESUMO
Pseudomonas aeruginosa RocR, an EAL-domain protein which regulates the expression of virulence genes and biofilm formation, has been cloned and expressed in Escherichia coli and purified. Here, the crystallization and preliminary diffraction analysis of RocR are reported. The X-ray diffraction data were processed to a resolution of 2.50 A. The crystals belonged to space group P6(1)22 or P6(5)22, with unit-cell parameters a = 118.8, b = 118.8, c = 495.1 A, alpha = beta = 90, gamma = 120 degrees .