ABSTRACT
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.
Subject(s)
5' Untranslated Regions/genetics , Genome, Viral , Phosphoproteins/chemistry , RNA, Viral , Tryptophan/chemistry , Vesiculovirus/chemistry , Viral Structural Proteins/chemistry , Amino Acid Sequence , Animals , Binding Sites , Chlorocebus aethiops , Hydrophobic and Hydrophilic Interactions , Kinetics , Models, Molecular , Molecular Chaperones , Molecular Sequence Data , Mutation , Phosphoproteins/genetics , Protein Binding , Protein Multimerization , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Spectrometry, Fluorescence , Tryptophan/genetics , Vero Cells , Vesiculovirus/genetics , Viral Structural Proteins/geneticsABSTRACT
The phosphoprotein (P protein) of Chandipura virus (CHPV), a negative stranded RNA virus, is involved in both transcription and replication phases of the viral life cycle. The two Tryptophan (Trp) residues of CHPV, located at 105 and 135 respectively and two single Trp mutants W135F and W105F and a double Trp mutant W135F/W105F have been characterized by steady state and time-resolved fluorescence and phosphorescence at 298 K and 77 K. Results indicate that Trp135 is more buried with less polar and more hydrophobic environment whereas the Trp105 is solvent exposed. Quantum yields (capital EF, Cyrillic) suggest that the singlet-singlet (S <--> S) non-radiative energy transfer (ET) from the Trp135 to the Trp105 occurs with 66% efficiency. The simulation of the fluorescence spectra of the WT and the time resolved studies support the results. Lifetime and capital EF, Cyrillic of the single Trp mutants suggest an intrinsic static quenching of the Trp105. The results at 77 K indicate that the ET takes place from the lowest triplet state (T(1)) of the Trp105 to the T(1) of the Trp135 apart from the backward S <--> S ET from the Trp105 to the Trp135. The triplet-triplet (T <--> T) ET implies a distance of <10 A between the Trp105 and the Trp135. Using the crystal structure of Vesicular Stomatitis Virus (VSV) phosphoprotein exhibiting about 34% similarity with the CHPV P protein, a homology modelling of CHPV supports the observed distance between the Trp residues, the S <--> S ET efficiency and the environments of the Trp residues in CHPV.
Subject(s)
Energy Transfer , Mutation , Phosphoproteins/chemistry , Tryptophan/genetics , Tryptophan/metabolism , Vesiculovirus , Viral Proteins/chemistry , Energy Transfer/radiation effects , Fluorescence Polarization , Models, Molecular , Mutagenesis, Site-Directed , Phosphoproteins/genetics , Phosphoproteins/metabolism , Protein Conformation , Sequence Homology, Amino Acid , Spectrometry, Fluorescence , Temperature , Viral Proteins/genetics , Viral Proteins/metabolismABSTRACT
We have investigated the organization and dynamics of tryptophan residues in tetrameric, monomeric and unfolded states of soybean agglutinin (SBA) by selective chemical modification, steady-state and time-resolved fluorescence, and phosphorescence. Oxidation with N-bromosuccinimide (NBS) modifies two tryptophans (Trp 60 and Trp 132) in tetramer, four (Trp 8, Trp 203 and previous two) in monomer, and all six (Trp 8, Trp 60, Trp 132, Trp 154, Trp 203 and Trp 226) in unfolded state. Utilizing wavelength-selective fluorescence approach, we have observed a red-edge excitation shift (REES) of 10 and 5 nm for tetramer and monomer, respectively. A more pronounced REES (21 nm) is observed after NBS oxidation. These results are supported by fluorescence anisotropy experiments. Acrylamide quenching shows the Stern-Volmer constant (K(SV)) for tetramer, monomer and unfolded SBA being 2.2, 5.0 and 14.6 M(-1), respectively. Time-resolved fluorescence studies exhibit biexponential decay with the mean lifetime increasing along tetramer (1.0 ns) to monomer (1.9 ns) to unfolded (3.6 ns). Phosphorescence studies at 77 K give more structured spectra, with two (0,0) bands at 408.6 (weak) and 413.2 nm for tetramer. However, a single (0,0) band appears at 411.8 and 407.2 nm for monomer and unfolded SBA, respectively. The exposure of hydrophobic surface in SBA monomer has been examined by 8-anilino-1-naphthalenesulfonate (ANS) binding, which shows approximately 20-fold increase in ANS fluorescence compared to that for tetramer. The mean lifetime of ANS also shows a large increase (12.0 ns) upon binding to monomer. These results may provide important insight into the role of tryptophans in the folding and association of SBA, and oligomeric proteins in general.
