RESUMO
The remarkable ability of baculovirus is to hyperexpress very late genes, but the mechanisms remain unclear. Here we report the effect of PK1, a baculovirus-encoded serine/threonine kinase, on very late gene hyperexpression. PK1 knockout does not completely disrupt very late gene expression, but down regulates the hyperexpression. Those truncated PK1s that exhibit kinase activity in vitro rescue the decline of very late hyperexpression, while other truncated PK1s and a point mutant PK1 (D137A) without kinase activity fail to rescue the decline of very late hyperexpression, suggesting that PK1 regulates very late gene expression by its kinase activity. In addition, those PK1 mutants that can rescue the hyperexpression are able to interact with very late promoter containing 5' UTR. Based on the above data, we hypothesize that PK1 binds to very late promoter containing 5' UTR to regulate the hyperexpression of very late genes by its kinase activity.
Assuntos
Regulação Enzimológica da Expressão Gênica/fisiologia , Regulação Viral da Expressão Gênica/fisiologia , Nucleopoliedrovírus/classificação , Nucleopoliedrovírus/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Virais/metabolismo , Animais , Nucleopoliedrovírus/genética , Proteínas Serina-Treonina Quinases/genética , Células Sf9 , Proteínas Virais/genética , Replicação Viral/fisiologiaRESUMO
PK-1 (Ac10) is a baculovirus-encoded serine/threonine kinase and its function is unclear. Our results showed that a pk-1 knockout AcMNPV failed to produce infectious progeny, while the pk-1 repair virus could rescue this defect. qPCR analysis demonstrated that pk-1 deletion did not affect viral DNA replication. Analysis of the repaired recombinants with truncated pk-1 mutants demonstrated that the catalytic domain of protein kinases of PK-1 was essential to viral infectivity. Moreover, those PK-1 mutants that could rescue the infectious BV production defect exhibited kinase activity in vitro. Therefore, it is suggested that the kinase activity of PK-1 is essential in regulating viral propagation. Electron microscopy revealed that pk-1 deletion affected the formation of normal nucleocapsids. Masses of electron-lucent tubular structures were present in cell transfected with pk-1 knockout bacmid. Therefore, PK-1 appears to phosphorylate some viral or cellular proteins that are essential for DNA packaging to regulate nucleocapsid assembly.
Assuntos
Nucleocapsídeo/metabolismo , Nucleopoliedrovírus/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Spodoptera/virologia , Montagem de Vírus , Animais , Células Cultivadas , Replicação do DNA , Regulação Viral da Expressão Gênica , Proteínas Serina-Treonina Quinases/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação ViralRESUMO
Rice stripe virus (RSV) belongs to the genus Tenuivirus and its genome consists of four single-stranded RNAs encoding seven proteins. Here, we have analyzed the processing and membrane association of Pc2 encoded by vcRNA2 in insect cells. The enhanced green fluorescent protein (eGFP) was fused to the Pc2 and used for the detection of Pc2 fusion proteins. The results showed that Pc2 was cleaved to produce two proteins named Pc2-N and Pc2-C. When expressed alone, either Pc2-N or Pc2-C could transport to the Endoplasmic reticulum (ER) membranes independently. Further mutagenesis studies revealed that Pc2 contained three ER-targeting domains. The results led us to propose a model for the topology of the Pc2 in which an internal signal peptide immediately followed a cleavage site, and two transmembrane regions are contained.
Assuntos
Tenuivirus/fisiologia , Proteínas Virais/metabolismo , Animais , Linhagem Celular , Retículo Endoplasmático/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Insetos , Processamento de Proteína Pós-Traducional , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Coloração e Rotulagem , Proteínas Virais/genéticaRESUMO
Rice stripe virus (RSV) infects rice and is transmitted in a propagative manner by the small brown planthopper. How RSV enters an insect cell to initiate the infection cycle is poorly understood. Sequence analysis revealed that the RSV NSvc2 protein was similar to the membrane glycoproteins of several members in the family Bunyaviridae and might induce cell membrane fusion. To conveniently study the membrane fusion activity of NSvc2, we constructed cell surface display vectors for expressing Nsvc2 on the insect cell surface as the membrane glycoproteins of the enveloped viruses. Our results showed that NSvc2 was successfully expressed and displayed on the surface of insect Sf9 cells. When induced by low pH, the membrane fusion was not observed in the cells that expressed NSvc2. Additionally, the membrane fusion was also not detected when co-expressing Nsvc2 and the viral capsid protein on insect cell surface. Thus, RSV NSvc2 is probably different from the phlebovirus counterparts, which could suggest different functions. RSV might enter insect cells other than by fusion with plasma or endosome membrane.
