Rinderpest virus (RPV) belongs to the order Mononegavirale which comprises non segmented negative sense RNA viruses including human pathogens such as Measles, Ebola and Marburg virus. RPV is the causative agent of Rinderpest disease in large ruminants, both domesticated and wild. The viral genome contains a non segmented negative sense RNA encapsidated by nucleocapsid protein (N-RNA). Viral transcription/replication is carried out by the virus encoded RNA dependent RNA polymerase represented by the large protein L and phosphoprotein P as (L-P) complex. Viral transcription begins at the 3’ end of the genome 3’le-N-P-M-F-H-N-tr-5’ with the synthesis of 55nt leader RNA followed by the synthesis of other viral mRNAs. A remarkable feature common to all members of Paramyxoviridae family is the gradient of transcription from 3’ end to the 5’ end of the genome due to attenuation of polymerase transcription at each gene junction. The present study aims at functional characterization of Rinderpest virus transcription and the associated activities required for viral mRNA capping. In addition, an attempt has been made to understand the novel role of a host factor, Ebp1, playing a key role in virus multiplication in infected cells. The specific aims of the study are presented in detail below. 1. Development of in vitro transcription system for RPV mRNA synthesis and role of phosphorylation of P protein in transcription. The transition of viral polymerase from transcription to replication in infected cells has been a long standing puzzle in all paramyxoviruses. Earlier work carried out using RPV minigenome with a CAT reporter gene and studies with phosphorylation null mutant P, has revealed the importance of P phosphorylation for viral transcription in vivo. However, the contribution of other cellular factors in the viral transcription/replication switch could not be ruled out in these assays. In order to understand the specific role of P protein in transcription/replication, it was necessary to develop a cell free transcription system for viral mRNA synthesis. Hence, viral genomic RNA (N-RNA) was purified from RPV infected cells using CsCl density gradient centrifugation. The viral RNA polymerase consisting of L-P complex was separately expressed in insect cells and partially purified by glycerol gradient centrifugation. Glycerol gradient fraction containing the L-P complex was found to be active in viral transcription. Notably, the gradient of transcription of viral mRNA was observed in vitro with the partially purified recombinant L-P complex similar to in vivo. However, the recombinant polymerase complex failed to synthesis the 55nt leader RNA, in agreement with the recent finding in VSV that the transcriptase complex was unable to synthesize leader RNA and viral transcription is initiated at the N gene start site unlike the conventional 3’ entry mode. The newly developed in vitro reconstituted transcription system was used to analyze the effect of P phosphorylation on viral transcription. The results presented in chapter 2, indicate that phosphorylated P supports transcription whereas unphosphorylated P transdominantly inhibits the transcription in vitro suggesting the possible role of the status of P protein phosphorylation in determining transcription/replication switch. 2. Enzymatic activities associated with RPV L protein- role in viral mRNA capping. Post transcriptional modification of mRNA such as capping and methylation determines the translatability of viral mRNA by cellular ribosome. In negative sense RNA viruses, synthesis of viral mRNA is carried out by the viral encoded RNA polymerase in the host cell cytoplasm. Since the host capping and methylation machinery is localized to the nucleus, viruses should either encode their own mRNA modification enzymes or adopt alternative methods as has been reported for orthomyxoviruses (cap snatching) and picornaviruses (presence of IRES element). In order to test, if RPV RNA polymerase possesses any of the capping and methylation activities, both virus as well as the RNP complex containing the viral N-RNA and RNA polymerase (L-P) were purified from infected cells. Using the purified virus and RNP complex, the first two activities required for mRNA capping vis-à-vis, RNA triphosphatase and guanylyltransferase were tested and the results are described in chapter 3 and 4. Purified virus as well as the RNP complex showed both RNA triphosphatase (RTPase) and Nucleotide triphosphatase activities. Neither purified N-RNA or recombinant P proteins show these activities suggesting that it is indeed mediated by viral L protein. By the metal dependency of the reaction and by the motif conservation with other reported RTPases, RPV L protein was assigned to the metal dependent RTPase tunnel family. Capping activity was also seen with the L protein present in RNP complex by its ability to form a covalent complex with GMP moiety of GTP. The specificity of the reaction with GTP, inhibition of Enzyme-GMP complex formation by the inorganic pyrophosphate and the susceptibility of Enzyme-GMP complex under acidic conditions clearly indicated that RPV L represents the viral guanylyl transferase. Further confirmation was obtained by the indirect capping assay in which Enzyme-GMP complex was formed when recombinant L protein was incubated with the cap labeled RNA due to the reversible nature of capping reaction. Owing to the large size of L protein (240 KDa), it is conceivable that the L protein functions in a modular fashion for different activities pertaining to RNA synthesis and modification. Sequence comparison of L proteins from different morbilliviruses revealed the presence of three conserved domains namely domain I (aa 1-606), domain II (aa 650-1694) and domain III (aa 1717-2183). Since domain II has already been assigned as the viral RNA dependent RNA polymerase, domain I and domain III were chosen for further characterization. Both domains were cloned, expressed and purified to homogeneity using recombinant baculovirus expression system. However, the recombinant domain III alone showed the NTPase activity where as neither domain I or III showed RTPase activity. This is expected since a part of the conserved RTPase motif was located in domain II in the multiple sequence alignment with other viral and yeast RTPases. In addition, the recombinant domain III also showed the characteristic enzyme-GMP complex formation but failed to be active in the indirect capping assay. Therefore, both domain II and domain III are likely to be involved in the co-transcriptional capping of viral mRNA. In support of this view, recent report in VSV suggests the presence of additional motif in domain II which is essential for viral mRNA capping. Preliminary evidence has been presented in the appendix section for the presence of N7 guanine methyl transferase activity with L protein although further experiments are needed to confirm this activity. 3. Role of host factor Ebp1 in negative sense RNA virus replication - a possible antagonist In recent years, many cellular factors such as actin, tubulin and profilin have been shown to be involved in viral transcription. Ebp1-ErbB3 binding protein was initially isolated as a cellular protein which binds to Influenza viral polymerase subunit PB1. Ebp1 selectively inhibits the influenza virus transcription in vitro whereas the cap binding and endonuclease activity of PB1 subunit of viral polymerase is unaffected. Till now there are no reports of the role of Ebp1 in non segmented negative sense RNA virus infection. The fifth chapter describes the role of Ebp1 in RPV infection and vice versa. RPV infection leads to down regulation of Ebp1 mRNA levels which in turn leads to decreased protein synthesis. Subsequently, it was found that Ebp1 interacts presumably with viral N protein, being a part of the viral RNP complex in both infected cells as well as in purified virion. Further, over expression of Ebp1 inhibits viral transcription and as a consequence the virus multiplication in vivo suggesting a mutual antagonism between virus and the host cell through Ebp1 protein.

Peste des petits ruminants virus (PPRV) belongs to the family paramyxoviridae which comprises non segmented negative sense RNA viruses including measles and rinderpest virus. PPRV is the causative agent of peste des petits rumaninats disease (also known as sheep or goat plague disease) in small ruminants. The viral genome contains a non segmented negative sense RNA encapsidated by viral encoded nucleocapsid protein (N-RNA). Viral transcription is carried out by the virus encoded RNA dependent RNA polymerase complex represented by the large protein L and phosphoprotein P. Viral transcription begins at the 3’ end of the genome synthesising all the viral transcripts (3’-N-P-M-F-HN-L-5’). A remarkable feature common to all members of Paramyxoviridae family is the gradient of transcription from 3’ end to the 5’ end due to attenuation of polymerase transcription at each gene junction. The objectives of the present study are characterization of peste des petits ruminants virus transcription and the associated activities required for post transcriptional modification of viral mRNA. In addition, an attempt has been made to develop in vitro transcription with heterologous combination of PPRV and RPV polymerase proteins. The first reaction in capping involves removal of γ-phosphate from triphosphate ended precursor mRNA by RNA triphosphatase. The domain having RNA triphosphatase activity within the L protein has been identified and expressed independently in E. coli. The details of the objectives are presented below. 1. Development of in vitro transcription system for PPRV mRNA synthesis In order to develop an in vitro transcription reconstitution system for PPRV, the viral RNP complex comprising large (L), phospho (P) and N protein encapsidating viral genomic RNA was purified from virus infected Vero cells. The in vitro transcription reconstituted system with RNP complex was able to synthesise all the viral mRNA as analysed by RT-PCR. As a control, total RNA from virus infected cells was isolated and analysed by RT-PCR. In order to refine the in vitro transcription system, separately expressed recombinant polymerase complex was used to reconstitute transcriptional activity in vitro. For this,viral genomic RNA (N-RNA) was purified from PPRV infected cells using CsCl density gradient centrifugation. The recombinant baculovirus for PPRV P protein was earlier generated in the lab. A recombinant baculovirus harbouring the L gene of PPRV was generated in the present study (described in part one). The viral RNA polymerase consisting of L-P complex was expressed in Sf21 insect cells and partially purified by ultra centrifugation on 5-20% glycerol gradient. Glycerol gradient fraction containing the L-P complex was found to be active in the in vitro transcription reconstitution system. Further quantitation of transcripts made in vitro and in infected cells has been carried out by real time PCR. Notably, the gradient of polarity of transcription of viral mRNA observed in vitro with the partially purified recombinant L-P complex was similar to the gradient observed in infected cells. Host proteins have been shown to modulate the transcription of many paramyxoviruses. In order to test the role of host factors, uninfected cell lysate of Vero cells was added to the in vitro transcription reaction and the transcript level was measured by real time PCR. The result showed an increase in the transcription by addition of host proteins suggesting the involvement of host factors in viral transcription. Further, the newly developed in vitro reconstitution system was used to test if recombinant L and P proteins of RPV can functionally replace PPRV L and P protein in the in vitro transcription complementation assay. The result presented in part one indicates that the L or P protein of PPRV can be replaced by RPV L and P protein in heterologous transcription reconstitution system ,with a reduced efficiency. However, the homologous polymerase complex of RPV failed to recognise the N-RNA genomic template of PPRV. 2. RNA triphosphatase activity of PPRV L protein and identification of RNA triphosphatase domain Post transcriptional modification of mRNA such as capping and methylation determines the translatability of viral mRNA by cellular ribosome. In negative sense RNA viruses, synthesis of viral mRNA is carried out by the viral encoded RNA polymerase in the host cell cytoplasm. Since the host capping and methylation machinery is localized to the nucleus, viruses should either encode their own mRNA modification enzymes or adopt alternative methods as has been reported for orthomyxoviruses (cap snatching) and picornaviruses (presence of IRES element). In order to test, if PPRV RNA polymerase possesses any of the capping activities, the RNP complex containing the viral N-RNA and RNA polymerase (L-P) were purified from virion. Using the purified RNP complex, the first activity required for mRNA capping, RNA triphosphatase was tested and the results are described in part two. RNP complex purified from virion showed both RNA triphosphatase (RTPase) activity. The RNA triphosphatase from viruses, fungi and other eukaryotes have been classified into two groups, metal dependent and metal independent. The cleavage of the γ-phosphate from triphosphate ended precursor mRNA by L protein of PPRV was found to be metal dependent. So, by the metal dependency of the RTPase reaction, PPRV L protein was assigned to the metal dependent RTPase tunnel family. One of the key features of metal dependent RTPase group members is the ability to hydrolyse γ-β phosphoanhydride bond of NTPs. PPRV L protein associated with RNP complex also was also able to cleave γ-β phosphoanhydride bond of NTPs. Owing to the large size of L protein (240 KDa), it is conceivable that the L protein functions in a modular fashion for different activities pertaining to mRNA synthesis and post transcriptional modification. Sequence comparison of L proteins from different morbilliviruses revealed the presence of three conserved domains namely domain I (aa 1-606), domain II (aa 650-1694) and domain III (aa 1717-2183). Domain II has the catalytic motif for viral RNA dependent RNA polymerase. Multiple sequence alignment of PPRV L protein with known RNA triphosphatases predicted a two hundred amino acid long region on L protein comprising the C terminus of domain II and N terminus of DIII as a possible candidate for RNA triphosphatase domain. The above predicted domain was cloned and expressed in E. coli. The ability of the purified recombinant RTPase domain to cleave γ-β phosphoanhydride bond of RNA was tested. The results described in part two suggest that the predicted RTPase domain has RNA triphosphatase activity. In addition to RNA triphosphatase, the RTPase domain also has the NTPase activity. The RNA triphosphatase of DNA viruses, yeasts and other fungi have three motifs essential for enzyme activity. Motif A and motif C are rich in glutamate and are involved in metal binding. Motif B is rich in basic amino acids and forms the centre for catalysis. The glutamate residue (E1647) of motif A of PPRV L protein RTPase domain was converted to alanine and the loss of RTPase activity was assessed. The results summarised in appendix 1 shows that the E1647A mutant has reduced RNA triphosphatase and NTPase activity.