Functional Characterization of C4 Protien of Cotton Leaf Curl Kokhran Virus - Dabawali

Abstract

1) Geminiviruses are a group of plant viruses which contain circular single stranded DNA molecules as their genomes and the capsid consists of two icosahedra fused together to form twinned or geminate particles. The largest genus in the family Geminiviridae is that of begomoviruses which are of two kinds; the monopartite begomoviruses which contain only one circular single stranded DNA molecule as their genome and the bipartite begomoviruses which contain two circular single stranded DNA molecules (designated DNA-A and DNA-B) as their genomes. In bipartite viruses, the two DNA molecules are enclosed in separate geminate capsids. 2) In bipartite begomoviruses, the DNA-A encodes the proteins essential for replication and encapsidation of the viral genome while the DNA-B encodes the proteins involved in movement. The DNA-B encodes two proteins: the BV1 or the nuclear shuttle protein (NSP) and BC1 or the cell-to-cell movement protein. Geminiviruses have DNA genomes which replicate inside the host cell nucleus. The NSP, which contains nuclear localization signal, brings the viral DNA from nucleus to the cytoplasm while the BC1 serves to take the viral genome to the cell periphery for movement to the neighbouring cell through the plasmodesmata. 3) The monopartite begomoviruses do not contain DNA-B (which, in bipartite begomoviruses, encodes the proteins involved in movement) and it has been suggested that some of the proteins encoded by DNA-A take up the movement function. Based on studies on TYLCV and CLCuV, a model has been proposed for the movement of monopartite begomoviruses according to which the coat protein (CP) of monopartite begomoviruses serves as the functional equivalent of the NSP of bipartite begomoviruses 4) The present thesis deals with the biochemical characterization of the C4 protein of the monopartite begomovirus CLCuKV-Dab. As stated in statement (3) above, the V2 and C4 proteins of monopartite begomoviruses have been implicated to be involved in cell-to¬cell movement of the viral genome. In TYLCV, both the proteins were shown to be localized to the cell periphery and could move from one cell to another through the plasmodesmata. Further, the V2 protein of CLCuKV-Dab was shown to interact with the coat protein and bind to single stranded DNA. The biochemical properties of the C4 protein needed to be elucidated in order to strengthen the proposal of its probable involvement in movement. 5) The objectives of the present study were: i) Bioinformatic analysis of the C4 protein of CLCuKV-Dab ii) Biochemical characterization of ATPase and pyrophosphatase activities of the C4 protein. iii) Studies on the effect of V2-C4 interaction on the enzymatic properties of C4. iv) Functional characterization of C4 in planta. 6) The FoldIndex© and PONDR analyses predicted the C4 protein of CLCuKV-Dab to be natively unfolded. Similarly, in PSIpred analysis, most of the C4 protein was predicted to be a random coil without any well-defined secondary structure. Further, the protein sequence was analyzed using the motifscan server. However, no motif for any specific function was predicted in the C4 Protein. 7) The C4 gene was initially cloned into pRSET-C vector and overexpressed as histidine tagged protein and the solubility of the protein was tested in various conditions including low temperature (18° C) after inducing the expression of the protein, buffers of various pH and different salt concentrations but the protein remained insoluble. Subsequently, the protein was purified under denaturing conditions and attempts were made to refold the protein but the protein precipitated during refolding. In order to get the C4 protein in soluble form, the C4 gene was subcloned into pGEX-5X2 vector and overexpressed as a GST-tagged fusion protein (GST-C4). Some of the GST-C4 protein was soluble which was purified by using GST-bind resin. The purified fusion protein was observed as a 37 kDa band on SDS-PAGE gel. The purified protein was accompanied by a degraded product of approximately 30 kDa size. Both the intact GST-C4 protein and the degraded product were detected in western blot analysis using anti-GST antibody. 8) Because C4 has been implicated to be involved in movement of monopartite begomoviruses and movement is an energy requiring process, it was of interest to determine if GST-C4 possesses ATPase activity. The purified GST-C4 protein was incubated with γ-[32P]-ATP, the product of the reaction was separated by thin layer chromatography and the chromatography plate was analyzed by phosphorimager. The hydrolysis of ATP by GST-C4 and the release of inorganic phosphate was clearly observed, suggesting that GST-C4 might possess ATPase activity. 9) The reaction conditions for the ATPase activity of GST-C4 were standardized. The activity increased linearly upto 2.60μM of the protein. The optimum temperature and pH for the ATPase activity were found to be 30 C and 6.0 respectively. The activity was inhibited by EDTA, suggesting that it is dependent on divalent metal ions. The activity was stimulated by Mg+2, Mn+2 and Zn+2 but inhibited by Ca+2ions. Further, in the time course experiment, it was observed that the ATPase activity increased linearly upto one hour. 10) The Km, Vmax and kcat for the ATPase activity of GST-C4 were found to be 51.72 ± 2.5 µM, 7.2 ± 0.54 nmoles/min/mg of the protein and 0.27 min-1 respectively. Some of the other virally encoded ATPases have been found to exhibit kcat similar to that found for GST-C4 but it is much lower than those of most of the prokaryotic and eukaryotic ATPases (as mentioned in Table 3.3, page 100, chapter 3). Further, the presence of the degraded product did not affect the kinetic constants as described in chapter 3, pages 95¬-98. It is possible that the enzymatic activity might increase upon interaction with some ligand. 11) In the absence of any putative ATP binding motifs, systematic deletions from N-and C-termini were made to delineate the regions of C4 important for the ATPase activity. GST-N∆15-C4 and GST-N∆30-C4 exhibited approximately 70 % reduction in the ATPase activity while all the C-terminal deletion mutants (GST-C∆10-C4, GST-C∆20¬C4 and GST-C∆30-C4) retained the activity similar to the full length GST-C4 protein. This suggested that the N-terminal region of C4 may contain the residues important for the ATPase activity of GST-C4. 12) In the N-terminal region of C4, there is a sequence CSSSSR which closely resembles the sequence present at the active site of phosphotyrosine phosphatases (CXXXXXR). However, GST-C4 did not catalyze the hydrolysis of p-Nitrophenyl phosphate, a substrate analogue commonly used to assay phosphotyrosine phosphatase activity. It was of interest to determine if the cysteine and arginine in this sequence are important for the ATPase activity of GST-C4. GST-R13A-C4 exhibited an approximately two fold reduction in Vmax suggesting that R13 in C4 may be catalytically important for the ATPase activity of GST-C4. On the other hand, the C8A mutation did not affect the ATPase activity of GST-C4. 13) The GST-C4 protein was tested for its ability to hydrolyze several other phosphate containing compounds as mentioned chapter 2, pages 53-55. Among these compounds, GST-C4 catalyzed the hydrolysis of sodium pyrophosphate, that is, GST-C4 exhibited an inorganic pyrophosphatase activity. 14) The reaction conditions for the inorganic pyrophosphatase activity of GST-C4 were initially standardized. The pyrophosphatase activity of GST-C4 increased linearly upto 3.38 µM of the protein. The optimum temperature and pH for the pyrophosphatase activity were found to be 37° C and 7.0 respectively. The pyrophosphatase activity was inhibited by EDTA, suggesting that it is dependent on divalent metal ions. The activity was most efficiently stimulated by Mg+2, although it was also stimulated by Mn+2and Zn+2but inhibited by Ca+2ions. Thus, the pyrophosphatase activity of GST-C4 resembles the family I inorganic pyrophosphatases in metal ion requirements. Further, the pyrophosphatase activity increased linearly upto 1 hour 30 minutes. 15) The Km, Vmax and Kcat for the pyrophosphatase activity of GST-C4 were found to be 0.76 ± 0.04 mM, 141.16 ± 20 nmoles/min/mg of the protein and 5.2 minrespectively. The kcat for the pyrophosphatase activity was approximately 20 fold higher than that for the ATPase activity (0.27 min-1). 16) GST-N∆15-C4 and GST-N∆30-C4 exhibited >70 % reduction in the pyrophosphatase activity, a finding similar to that for the ATPase activity. On the other hand, while GST-C∆10-C4 retained the activity similar to the full length GST-C4 protein, GST-C∆20-C4 and GST-C∆30-C4 exhibited 20 % and 60 % reduction in the pyrophosphatase activity, respectively, as compared to the full length GST-C4 protein. This suggested that the C-terminal region of C4 may also contain the residues important for the pyrophosphatase activity of GST-C4. However, the C-terminal deletion mutants retained the ATPase activity similar to the full length protein. 17) The pyrophosphatase activity of GST-C4 was stimulated more than three fold by several reducing agents. The C4 protein contains only one cysteine (at position 8 in the C4 sequence). This was the first clue that the cysteine may be important for the pyrophosphatase activity of the GST-C4 protein. Further, the pyrophosphatase activity of GST-C4 did not exhibit preference for a particular kind of reducing agent like that of the pyrophosphatase activity in Streptococcus faecalis. 18) GST-C8A-C4 exhibited more than two fold reduction in Vmax, suggesting that the C8 may be catalytically important for the pyrophosphatase activity of GST-C4. On the other hand, the R13A mutation did not affect the pyrophosphatase activity of the GST-C4 protein. Thus, it is possible that during catalysis, the cysteine thiolate of C4 makes a 19) The pyrophosphatase activity of GST-C4 was inhibited by vanadate and fluoride. Vanadate was found to be a competitive inhibitor with Ki 0.33 mM while fluoride was a non-competitive inhibitor with Ki 2.82 mM. A comparative account of the two enzymatic activities of GST-C4 is presented in table 6.1