Systematic deletion and site-directed mutagenesis of FHVB2 establish the role of C-terminal amino acid residues in RNAi suppression.
Viruses and siRNA/miRNA machinery of the host cell interact in diverse ways with the virus encoded RNAi suppressor proteins. These interactions have implications on the replication and pathogenicity of the virus and also on the immune response of the host. Suppressor protein B2 of insect Flock House Virus (FHVB2), has been shown to mediate RNAi suppression via N-terminal region by directly binding to dsRNA. We have previously shown that FHVB2 protein also interacts with host Dicer protein via its PAZ domain. In the present study, we performed systematic mutagenesis studies to map the FHVB2 regions involved in mediating suppression of RNAi. Progressive deletion of 17 amino acids from N- and C-terminii of FHVB2 resulted in cumulative decrease in RNAi suppression activity of FHVB2. The deletion of 17 amino acids from the C-terminus resulted in more reduction in RNAi suppression in comparison to the N-terminal deletions. Subsequently, we generated 17 successive point mutants of FHVB2 C-terminus and evaluated the RNAi suppression activity for each of the point mutants. Each point mutation resulted in a significant reduction in RNAi suppression activity of FHVB2. These results provide evidence for the role of C-terminal of FHVB2 in RNAi suppression.
Singh
Gatikrushna
G
International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India.
Korde
Reshma
R
Malhotra
Pawan
P
Mukherjee
Sunil
S
Bhatnagar
Raj K
RK
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D030381:Nodaviridae / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference; D017423:Sequence Analysis, RNA; D017384:Sequence Deletion; D013489:Suppression, Genetic; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism
D014764:Viral Proteins