Functional characterization of VP1 of Infectious Bursal Disease Virus

VP1 is an internal polypeptide encoded by segment B of Infectious Bursal Disease Virus (IBDV), which is believed to have RNA dependent RNA polymerase and guanylyl-transferase activities. In order to characterize its functions, experiments were conducted towards expression of VP1 in vitro in a cell-free system. Recombinant plasmids, containing the complete open reading frame of VP1 of IBDV strain OH were transcribed using "Ambion MEGAscript in vitro Transcription Kit". Time course experiments, nucleotide concentration and plasmid DNA concentration optimizations were done to obtain the maximum transcription yield. Recombinant VP1 was expressed in vitro by using the rabbit reticulocyte lysate system. Transcripts concentration optimization reactions were also done to obtain maximum translation yield.

The recombinant VP1 obtained in cell-free system was further used for assaying the functional activities of VP1. However, no activity could be demonstrated in the recombinant VP1, even after various optimization reactions. To determine whether VP1 has a guanylyl-transferase activity "competition and replacement reactions" with cold and labeled GTP were performed. It was found that even though there was guanylylation of IBDV VP1, this VP1-GMP complex was not reversible and therefore VP1 could not transfer the labeled GMP to an acceptor molecule. Therefore, IBDV VP1 is not a guanylyl-transferase/capping enzyme.

In conclusion, the results of this study indicate that maximum transcription yield is obtained after 4 hours of incubation at 37.5mM nucleotide concentration. Recombinant VPI of approximately 80 kDa was obtained on analysing by SDS-PAGE followed by autoradiography. Efforts to detect for VP1 enzymatic activities utilizing recombinant VP1 were not successful. However, guanylylation activity associated with IBDV VP1 was characterized in detail by utilizing purified IBDV particles. (Abstract shortened by UMI.).