RNA-dependent RNA polymerase activity in two morphologically different white clover cryptic viruses.

Area of Science:

• Plant virology
• Molecular biology
• Enzymology

Background:

• Cryptic viruses are RNA viruses that infect plants.
• RNA-dependent RNA polymerase (RdRp) is essential for the replication of RNA viruses.

Purpose of the Study:

• To investigate the presence and characteristics of RNA-dependent RNA polymerase (RdRp) activity in purified particles of white clover cryptic viruses 1 and 2.
• To determine the optimal conditions and products of the viral RdRp activity.

Main Methods:

• Purification of white clover cryptic viruses 1 and 2.
• Assay of RNA-dependent RNA polymerase activity in vitro.
• Characterization of reaction products using CF 11 column chromatography, RNase A, and S1 nuclease digestion.
• Analysis of synthesized double-stranded RNAs (dsRNAs) via gel electrophoresis.

Main Results:

• RNA-dependent RNA polymerase (RdRp) activity was detected in purified particles of both white clover cryptic viruses 1 and 2.
• The optimal activity requirements for the RdRp of each virus differed.
• Enzyme activity required virus particles, Mg2+, and all four ribonucleoside triphosphates.
• Synthesized products were confirmed as double-stranded RNAs (dsRNAs) with electrophoretic mobilities identical to viral templates.
• The RdRp activity was insensitive to actinomycin D, alpha-amanitin, and rifampicin.

Conclusions:

• White clover cryptic viruses 1 and 2 possess intrinsic RNA-dependent RNA polymerase (RdRp) activity within their purified particles.
• The characterization of these viral RdRps provides insights into their replication mechanisms.
• The distinct optimal activity requirements suggest potential differences in the RdRp enzymes of the two cryptic viruses.