Peanut Clump Virus RNA-1-Encoded P15 Regulates Viral RNA Accumulation but Is Not Abundant at Viral RNA Replication Sites
| Autor: | C. Fritsch, Christophe Ritzenthaler, Etienne Herzog, Patrice Dunoyer, Odile Hemmer |
|---|---|
| Rok vydání: | 2001 |
| Předmět: |
Gene Expression Regulation
Viral Arachis Recombinant Fusion Proteins Immunology Replication Virus Replication Microbiology Plant Viruses Green fluorescent protein Viral Proteins Capsid Virology Plant virus Tobacco RNA Viruses Replicon Subgenomic mRNA Microscopy Confocal biology Protoplasts RNA Peanut clump virus biology.organism_classification Molecular biology Plants Toxic Microscopy Fluorescence Viral replication Cytoplasm Insect Science Mutation RNA Viral Subcellular Fractions |
| Zdroj: | Journal of Virology. 75:1941-1948 |
| ISSN: | 1098-5514 0022-538X |
| DOI: | 10.1128/jvi.75.4.1941-1948.2001 |
| Popis: | RNA-1 of peanut clump pecluvirus (PCV) encodes N-terminally overlapping proteins which contain helicase-like (P131) and polymerase-like (P191) domains and is able to replicate in the absence of RNA-2 in protoplasts of tobacco BY-2 cells. RNA-1 also encodes P15, which is expressed via a subgenomic RNA. To investigate the role of P15, we analyzed RNA accumulation in tobacco BY-2 protoplasts inoculated with RNA-1 containing mutations in P15. For all the mutants, the amount of progeny RNA-1 produced was significantly lower than that obtained for wild-type RNA-1. If RNA-2 was included in the inoculum, the accumulation of both progeny RNAs was diminished, but near-normal yields of both could be recovered if the inoculum was supplemented with a small, chimeric viral replicon expressing P15, demonstrating that P15 has an effect on viral RNA accumulation. To further analyze the role of P15, transcripts were produced expressing P15 fused to enhanced green fluorescent protein (EGFP). Following inoculation to protoplasts, epifluorescence microscopy revealed that P15 accumulated as spots around the nucleus and in the cytoplasm. Intracellular sites of viral RNA synthesis were visualized by laser scanning confocal microscopy of infected protoplasts labeled with 5-bromouridine 5′-triphosphate (BrUTP). BrUTP labeling also occured in spots distributed within the cytoplasm and around the nucleus. However, the BrUTP-labeled RNA and EGFP/P15 very rarely colocalized, suggesting that P15 does not act primarily at sites of viral replication but intervenes indirectly to control viral accumulation levels. |
| Databáze: | OpenAIRE |
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