Browsing by Author "Dutta, Pinky"

The trans-replication system of a virus deals with the idea where the viral RNA involved in the translation of replicase protein is separate from the one involved in the replication of the virus. This system has been successfully used to study virus replication complexes and numerous viral and host factors involved in the replication and infection process of Semliki Forest virus, Chikungunya virus and Sindbis virus. We attempted to test for the feasibility of this system with Potato virus X (PVX), an alpha-like virus, in planta. A viral RNA template was designed to produce a non-functional replicase protein, with the coat protein sequence deleted to prevent formation of virions. All other RNA features of the template sequence had been left unaltered, possibly making it recognizable by the replicase. The replicase construct encodes for the replicase protein and its RNA lacks other virus-specific recognition sequences. Both of the constructs were delivered into the mesophyll cells of the Nicotiana benthamiana leaves via Agrobacterium-mediated infiltration. Templates of various lengths, ranging from 2569 to 7562 nucleotides were tested. The longer templates did not replicate at 4 and 6 days post inoculation, when the replicase protein was provided in trans. Further optimization of the system with shorter templates and addition of helper component proteinase (HCPro), a potyviral silencing suppressor, led to effective trans-replication of the templates in plant cells. However, the replication sites were observed to be scattered across the leaf lamina suggesting that further optimization is required for increased efficiency of the trans-replication system. All in all, it has been established that PVX is capable of trans-replicating and the experimental freedom offered by this system can be utilized to delve deeper into understanding the replication mechanism of the virus.