Interaction of the intrinsically disordered C-terminal domain of the sesbania mosaic virus RNA-dependent RNA polymerase with the viral protein P10 in vitro: modulation of the oligomeric state and polymerase activity
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The RNA-dependent RNA polymerase (RdRp) of sesbania mosaic virus (SeMV) was previously shown to interact with the viral protein P10, which led to enhanced polymerase activity. In the present investigation, the equilibrium dissociation constant for the interaction between the two proteins was determined to be 0.09 µM using surface plasmon resonance, and the disordered C-terminal domain of RdRp was shown to be essential for binding to P10. The association with P10 brought about a change in the oligomeric state of RdRp, resulting in reduced aggregation and increased polymerase activity. Interestingly, unlike the wild-type RdRp, C-terminal deletion mutants (C del 43 and C del 72) were found to exist predominantly as monomers and were as active as the RdRp-P10 complex. Thus, either the deletion of the C-terminal disordered domain or its masking by binding to P10 results in the activation of polymerase activity. Further, deletion of the C-terminal 85 residues of RdRp resulted in complete loss of activity. Mutation of a conserved tyrosine (RdRp Y480) within motif E, located between 72 and 85 residues from the C-terminus of RdRp, rendered the protein inactive, demonstrating the importance of motif E in RNA synthesis in vitro.
We thank the Department of Biotechnology (DBT), India (Grant ID DBT/BF/PRINS/2011-12/IISc): the Department of Science and Technology (DST), India (Grant ID No. SR/S2/JCB-60/2007); and the Indian Institute of Science, India, for financial support. HSS acknowledges the DST for a J C Bose fellowship, and the Indian National Science Academy for support. AB acknowledges the Council of Scientific and Industrial Research (CSIR), India, for a Senior Research Fellowship. We thank Dr. Govind for providing the pET 22b and pRSF Duet constructs and for his guidance during the initial experiments.
The study was funded by the Department of Biotechnology (DBT), India (Grant ID DBT/BF/PRINS/2011-12/IISc) and the Department of Science and Technology (DST), India (Grant ID No. SR/S2/JCB-60/2007).
Compliance with ethical standards
Conflict of interest
All of the authors (Arindam Bakshi, Shruthi Sridhar, Srinivas Sistla and Handanahal Subbarao Savithri) declare that he/she has no conflict of interest regarding the work carried out in the article.
This article does not contain any studies with human participants or animals performed by any of the authors.
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