Abstract In malignancies many alterations involving matrix macromolecule synthesis, secretion and assembly into basement membranes (BMs) as well as their degradation are present. The most important groups associated with matrix turnover are matrix metalloproteinases (MMPs) and their inhibitors (TIMPs). In this study altogether 285 tissue samples were investigated comprising various malignant epithelial tumors and normal tissue structures, in which the distribution of different laminin chains was studied immunohistochemically. Laminin α5, β1 and γ1 were detected almost in all the BMs studied including normal tissues and malignancies, whereas α1 chain of laminin was present only in certain BMs. Laminin γ2 chain was solely expressed by epithelial BMs and was present in intracellular space especially in individual carcinoma cells infiltrating in the tumor stroma and in tumor cells in close contact with BM zone. Generally epithelial tumors contained quite well-formed BMs around their tumor clusters, except for infiltrative breast carcinoma and diffuse type gastric carcinoma. In situ hybridization revealed that only epithelial cells contained mRNAs for laminin α1 and γ2 chains, whereas laminin β1 chain and α1(IV) collagen were synthesized mainly by stromal cells. mRNA for MMP-2 was produced mainly by stromal cells in hepatocellular carcinoma of liver (HCC) and pancreatic adenocarcinoma, whereas MMP-9 and MT1-MMP were equally synthesized by carcinoma cells and cells of tumor stroma. However, in HCCs of grade III carcinoma cells predominated in their MT1-MMP expression. All three MMPs were immunolocalized to malignant epithelial cells and showed variably stromal cell positivity. Statistically mRNA synthesis for MT1-MMP was significantly associated with the shortened survival of patients with HCC (P ≤ 0.01). TIMP-1-3 mRNA, and especially TIMP-3, expressions in normal endometrium were significantly increased in endometrial stromal cells towards the secretory phase. In various endometrial hyperplasias TIMPs and MT1-MMP expressions were quite comparable to those seen in proliferating endometrium. In endometrial adenocarcinomas their expressions were significantly increased and the most intensified mRNA expressions were seen in grade III adenocarcinomas. Especially TIMP-3 and MT1-MMP mRNAs were synthesized by carcinoma cells. The results indicate that epithelial malignancies are capable of active synthesis and assembly of BM macromolecules. Simultaneous matrix synthesis and degradation seen in malignancies suggest that the mechanisms involved in matrix turnover are not lost during malignant transformation. mRNA synthesis for MMPs and TIMPs is generally increased in epithelial malignancies. The results therefore strongly support the concept that MMPs have an active role in carcinoma cell invasion.

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.