RESUMEN
To study subdomain organization of the potato virus X (PVX) movement protein (MP) encoded by the first gene in the triple gene block (TGB), we mutated the 25-kDa TGBp1 protein. The N-terminal deletion of the helicase motifs I, IA, and II resulted in loss of the ATPase activity and RNA binding. A frameshift mutation truncating the C-terminal motifs V and VI gave rise to increase of the TGBp1 ATPase activity and had little effect on RNA binding in vitro. Fusions of the green fluorescent protein with 25-kDa MP and its derivative lacking motifs V-VI exhibited similar fluorescence patterns in epidermal cells of Nicotiana benthamiana leaves. Cell-to-cell movement of the 25K-deficient PVX genome was not complemented by the TGBp1 of Plantago asiatica mosaic potexvirus (PlAMV) but was efficiently complemented by a chimeric TGBp1 consisting of the N-terminal part of PlAMV protein (motifs I-IV) and the PVX-specific C-terminal part (motifs V-VI). These results suggest that NTP hydrolysis, RNA binding, and targeting to the specific cellular compartment(s) are associated with the N-terminal domain of the TGBp1 including the helicase motifs I-IV and that the C-terminal domain is involved in specific interactions with other virus proteins.
Asunto(s)
Potexvirus , Proteínas Virales/fisiología , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , ADN Helicasas/metabolismo , Mutación del Sistema de Lectura , Prueba de Complementación Genética , Datos de Secuencia Molecular , Proteínas de Movimiento Viral en Plantas , Potexvirus/genética , ARN/metabolismo , Relación Estructura-Actividad , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
The 25K movement protein (MP) of potato virus X (PVX) is encoded by the 5'-proximal gene of three overlapping MP genes forming a 'triple gene block'. The PVX 25K MP (putative NTPase-helicase) has been synthesized in Escherichia coli as a recombinant containing a six-histidine tag at the amino terminus. The His-tagged 25K protein was purified in a one-column Ni-chelate affinity chromatography procedure. In the absence of any other viral factors, this protein had obvious Mg2+-dependent ATPase activity, which was stimulated slightly (1.7-1.9-fold) by various polynucleotides. Like other viral proteins possessing ATPase-helicase motifs and many plant viral movement proteins, the PVX 25K MP was able to bind nucleic acids in vitro. The RNA binding activity of the 25K MP was pronounced only at very low salt concentrations and was independent of its ATPase activity.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Potexvirus , Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Proteínas Virales/metabolismo , Ácido Anhídrido Hidrolasas/metabolismo , Secuencia de Aminoácidos , Escherichia coli/genética , Datos de Secuencia Molecular , Peso Molecular , Nucleósido-Trifosfatasa , Proteínas de Movimiento Viral en Plantas , ARN Helicasas , ARN Nucleotidiltransferasas/metabolismo , ARN Viral/farmacología , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/aislamiento & purificaciónRESUMEN
A simple new method for preparing plasmid DNA and preformed zwitterionic liposome complexes is proposed. The ability of these metallonucleoliposome complexes to serve as a vehicle for gene delivery to mammalian cells in vivo was studied. A high level of expression of the reporter gene introduced was observed in mouse skeletal muscles in vivo.