Characterization of the chandipura virus leader RNA-phosphoprotein interaction using single tryptophan mutants and its detection in viral infected cells.
The phosphoprotein (P protein) of the chandipura virus (CHPV), a negative strand RNA virus, is involved in both transcription and replication of the viral life cycle. Interaction between the P protein and the viral leader (le) RNA under in vitro conditions has been previously reported for CHPV and other negative strand RNA viruses such as the rinderpest virus (RPV). However, till date, the region of the P protein involved in le RNA binding remains undefined. Moreover, the in vivo occurrence of this interaction has not been studied before. Here, we have characterised the P protein-le RNA interaction, using single tryptophan mutants of the P protein. The CHPV P protein contains two tryptophan residues located at amino acid position 105 and 135 respectively. Our previous study showed that Trp 135 is located in a buried region within a less polar environment whereas Trp 105 is more solvent-exposed. In this study we have used steady state and time resolved fluorescence spectroscopy at 298 K to show that the buried tryptophan (Trp 135) is involved in the interaction with the le RNA and the more solvent exposed Trp 105 is only slightly perturbed during this interaction. We also show that Trp 135 is responsible for the dimerization of the CHPV P protein. In addition, we have been able to demonstrate for the first time that the P protein-le RNA interaction is detectable in CHPV-infected Vero-76 cells and this interaction is augmented during the replication phase of the viral cycle.
Dr. B. C. Guha Centre for Generic Engineering and Biotechnology, Department of Biotechnology, University of Calcutta, 35, Ballygunge Circular Road, Kolkata 700019, India.
Arunava
A
Roy
Manini
M
Mukherjee
Subhradip
S
Mukhopadhyay
Shyam S
SS
Maity
Sanjib
S
Ghosh
Dhrubajyoti
D
Chattopadhyay
D020121:5' Untranslated Regions / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002522:Chlorocebus aethiops; D016679:Genome, Viral; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008958:Models, Molecular; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D009154:Mutation; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics; D011485:Protein Binding; D055503:Protein Multimerization; D012367:RNA, Viral; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry* / Q000235:genetics; D014709:Vero Cells; D018116:Vesiculovirus / Q000737:chemistry* / Q000235:genetics; D015678:Viral Structural Proteins / Q000737:chemistry* / Q000235:genetics
D020121:5' Untranslated Regions; D018832:Molecular Chaperones; C412837:P protein, Chandipura virus; D010750:Phosphoproteins; D012367:RNA, Viral; D011994:Recombinant Proteins; D015678:Viral Structural Proteins; D014364:Tryptophan