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These (agrégé de l'enseignement supérieur)--Catholic University of Louvain. / At head of title: From the Rega Institute for Medical Research, the Catholic University of Louvain, Faculty of Medicine, Dept. of Virology. Summaries in English, Dutch, and French. Includes bibliographical references.

These (agrégé de l'enseignement supérieur)--Catholic University of Louvain. / At head of title: From the Rega Institute for Medical Research, the Catholic University of Louvain, Faculty of Medicine, Dept. of Virology. Summaries in English, Dutch, and French. Includes bibliographical references.

The influenza virus causes a disease that kills approximately 500,000 people worldwide each year. Influenza is a negative-sense RNA virus that encodes its own RNA-dependent RNA polymerase. This protein (FluPol) carries out both genome replication and viral transcription. Therefore, like the L-proteins of non-segmented negative-sense RNA (nsRNA) viruses, FluPol also contains mRNA capping and polyadenylation functionality. In FluPol, capping is achieved by snatching cap structures from cellular mRNAs, so requiring cap-binding and endonuclease activities. This makes FluPol a substantial machine. It is a heterotrimeric complex, composed of PB1, PB2 and PA/P3 subunits, with a total molecular weight of 255 kDa. PB1 houses the polymerase active site, whereas PB2 and PA contain, respectively, cap-binding and endonuclease domains. Currently, we only have high resolution structural information for isolated fragments of FluPol. This severely hampers our understanding of influenza replication and consequently inhibits the development of therapies against the virus. In this DPhil project, I have determined a preliminary structure for the heterotrimeric FluPol of influenza C/Johannesburg/1/66, solved by x-ray crystallography to 3.6 Å. Overall, FluPol has an elongated structure with a conspicuous deep groove. PB1 displays the canonical right-hand-like polymerase fold. It sits at the centre of the particle, sandwiched between the two domains of P3, and with PB2 stacked against one side of this dimer. In the structure, the polymerase and endonuclease catalytic sites are both ~40 Å away from the cap-binding pocket. This pocket also faces a tunnel leading to the polymerase core. This suggests a mechanism for how capped cellular mRNAs are cleaved and then fed into the polymerase active site to prime transcription. The structure also hints at a unique trajectory for template RNA, in which the RNA exits at an angle ~180° from which it came in. This provides an explanation for how the polymerases of influenza, and other nsRNA viruses, can copy templates that are packaged into ribonucleoprotein complexes. My work reveals the first molecular structure of any polymerase from an nsRNA virus. It uncovers the arrangement of functional domains within FluPol, illuminating the mechanisms of this and related viral polymerases. This work will help focus future experiments into FluPol biology, and should hopefully spur the development of novel antiviral drugs.

Viruses are submicroscopic obligate parasites and depend on the host cell for their growth and reproduction. Plants are infected by diverse group of viruses that mostly possess RNA as their genome. As exception, viruses belonging to the family Geminiviridae are DNA viruses and infect both mono and dicotyledonous plants causing a large economic loss. These viruses are smaller in size encoding fewer proteins and employ the host cell machinery for successful infection and spread. Geminiviruses undergo frequent recombinations due to mixed infection resulting in vast diversity across the family and account for driving evolution in these viruses. Movement in these viruses is complex since they have to cross two important barriers, nuclear and cell wall barrier to establish systemic spread. All these factors play very important role while designing control measures against these viruses. Thus a detailed understanding of these processes at molecular level is essential. Cotton is the major cash crop in Indian subcontinent with huge export values. India has become the second largest producer of cotton in the world. However, the major constraint in cotton cultivation has been crop loss due to diseases caused by viruses, particularly the cotton leaf curl disease (CLCuD) caused by begomoviruses. Present thesis deals with the analysis of genetic variability of CLCuD in India and functional characterization of proteins involved in the movement of Cotton Leaf Curl Kokhran Virus-Dabawali (CLCuKV-Dab). CLCuKV-Dab belongs to family Geminiviridae and subgroup Begomovirus. A review of the literature on Geminiviridae classification, genome organization, virus entry, replication, transcription, translation, assembly and movement is presented in Chapter 1. This chapter also includes the review of host factors involved in replication, geminiviral proteins involved in gene silencing and a detailed report on CLCuD complexes and sub viral DNAs that are associated with CLCuD. The materials used in this study and the experimental protocols followed such as construction of recombinant clones, their overexpression in both bacterial and baculovirus expression systems, Protein purification techniques, site directed mutagenesis and all other biochemical, molecular biology and cell biology methods are described in detail in Chapter 2. Previous study has reported the complete genomic sequences of CLCuKV-Dab and Tomato leaf curl Bangalore virus-cotton [Fatehabad] (ToLCBV-Cotton [Fat]) and partial sequence of CLCuKV-Gang and the Cotton leaf curl Rajasthan virus (CLCuRV-Ban). Phylogenetic analysis of DNA-A sequences of these viruses with other CLCuD causing viruses is discussed in detail in Chapter 3. Chapter 4 deals with overexpression, purification and functional characterization of CLCuKV-Dab CP in terms of its interaction with DNA, the kinetics and its role in cell to cell movement. The proposed partners to CP in the cell to cell movement of monopartite begomoviruses are AV2 and AC4. Thus the Chapter 5 describes the functional characterization of recombinant AV2 of CLCuKV-Dab. Chapter 6 deals with expression of CP and AV2 as GFP fusion proteins in insect cells using baculovirus expression system to study the localization patterns of these proteins. Chapter 7 describes functional characterization of CLCuKV-Dab AC4. Bioinformatic analysis of AC4 showed that it belongs to the rare group of natively unfolded proteins that are functionally active In conclusion, there is a large genetic variability that exists among the begomoviruses and in particular, among the CLCuD causing begomoviruses in India. Functional characterization of the proteins involved in the cell to cell movement in CLCuKV-Dab led to a possible model for its movement; the CP translated in the cytoplasm is targeted the nucleus via its NLS and there binds to progeny ssDNA and exports the ssDNA out of nucleus through its export signals. AC4 or some other host proteins yet to be identified transports the ssDNA-CP complex from the nuclear periphery to AV2 present at the cell periphery. The complex is then transported from one cell to the neighboring cell via plasmodesmata. AC4 being an ATPase/NTPase could provide energy for the process.

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