Characterization of the infectivity of Bamboo mosaic virus with its correlation to the in vitro replicase activities in Nicotiana benthamiana

Abstract

In vitro RNA-dependent RNA polymerase (RdRp) transcription assay share an extremely useful system for studying the molecular mechanisms of replication of positive-sense RNA viruses such as Bamboo mosaic virus (BaMV). However, the obstacle encountered in this system is the inconsistency in the enzyme activity and the template specificity among different batches of the RdRp extracts. In order to overcome this obstacle, we designed experiments to study the functional dynamics of the BaMV RdRp in terms of its activity and specificity during the course of infection. Several different batches of RdRp preparations, extracted from inoculated leaves at a different time intervals of post-inoculation, were tested for their in vitro RdRp transcription activities. Results of RdRp assays using endogenous templates showed that the transcription activity giving rise to the 6.4 kb genomic RNA reached a maximum at 5th dpi. The RdRp extracted at 5th dpi could differentiate between BaMV and CMV exogenous templates. Results of exogenous RNA template activities using the 3′-ends of plus- and minus-strand RNA indicated that the 5th dpi RdRp could initiate minus-strand RNA synthesis more efficiency than the 11th dpi RdRp.