IISc Logo    Title

etd AT Indian Institute of Science >
Division of Biological Sciences >
Microbiology and Cell Biology (mcbl) >

Please use this identifier to cite or link to this item: http://hdl.handle.net/2005/382

Title: Role Of Matrix Protein Of Rinderpest Virus In Viral Morphogenesis
Authors: Subhashri, R
Advisors: Shaila, M S
Keywords: Glycoproteins
Lipid Membranes
Viral Morphogenesis
Rinderpest Virus
Virus Solubilization
Viral Proteins
Cattle Plague
Matrix Protein
Submitted Date: Aug-2006
Abstract: Rinderpest virus is an enveloped Nonsegmented Negative Stranded RNA Virus (NNSV) belonging to the genus Morbillivirus in the Family Paramyxoviridae and the causative organism for “cattle plague”. The virion has a transport component and a replication component. The transport component consists of a lipid membrane with two external membrane-anchored glycoproteins, namely Hemagglutinin (H) and Fusion (F) proteins that are necessary for cell entry and release of newly formed virus particles. The replication component consists of viral genomic RNA encapsidated by the nucleoprotein (N) and a RNA polymerase complex (Large subunit L and phosphoprotein P). These two components are linked together by the matrix protein (M) that is believed to play a crucial role in the assembly and maturation of the virion particle by bringing the two major viral components together at the budding site in the host cell. To perform this function, M protein should be able to interact with the host cellular membrane, especially the plasma membrane in the case of Rinderpest virus, should be able to interact with itself to form multimers as well as with the nucleocapsid core. The function might include the interaction of M protein with the cytoplasmic tail of the other two envelope proteins namely F and H. To understand the role of matrix protein in Rinderpest virus life cycle, the following functions were characterized – 1) Matrix protein association with the host cell membrane. 2) Matrix protein association with nucleocapsid protein. Matrix protein association cellular membranes in rinderpest virus infected cells could be a result of its interaction with the cytoplasmic tails of the viral glycoproteins. Hence, this association was characterized in the absence of other viral proteins. In transiently transfected cells, M protein existed in two isoforms namely the soluble cytosolic form and membrane-bound form. The membrane-bound M protein associated stably with the membranes, most likely by a combination of electrostatic and hydrophobic interactions, which is inhibited at high salt or high pH, but not completely. Confocal microscopy analysis showed the presence of M protein in plasma membrane protrusions. When GFP was tagged with this protein, GFP was absent from nucleus and was present predominantly in the cytosol and the plasma membrane protrusions. However, M protein expression did not result in the release of membrane vesicles (Virus-like particles) into the culture supernatant implicating the requirement of other viral proteins in envelope acquisition. Matrix protein of RPV has been shown to co-sediment with nucleocapsid during mild preparation of RNP from virus-infected cells. This association was further investigated by virus solubilization. The matrix protein could be solubilised completely from virion only in the presence of detergent and high salt. This is in agreement with the previous observation from the laboratory that the purified matrix protein remained soluble in the presence of detergent and 1M NaCl. This suggested that M protein could oligomerise or associate with nucleocapsid. The purified M protein when visualized by Electron microscopy showed the presence of globular structures, which may be due to self association of M protein, which may be due to self-aggregation of M protein. The presence of GFPM in filamentous structures in transfected cells, as visualized by confocal microscopy could also be due to self-assembly of M protein. Interaction of matrix protein RPV nucleocapsid was confirmed using co- sedimentation and floatation gradient analysis. Results obtained from M-N binding assay using C-terminal deletions of nucleocapsid protein suggested that the matrix protein interacted with the conserved N-terminal core of nucleocapsid and non- conserved C-terminus 20% is dispensable. This is in agreement with the report that RPV M protein could be replaced with that of Peste-des-petits-ruminants virus(a closely related morbillivirus). The observation that the nucleocapsid protein interacts with both soluble and membrane-bound form suggests that the matrix protein can possibly interact itself to facilitate the assembly of replication component at the site of budding where the transport component is already assembled. Viral proteins of many RNA viruses interact with detergent-resistant host components that facilitate their transport inside the cell to the sits of assembly or replication. Rinderpest viral proteins acquire detergent resistance in infected cells. This acquisition is mediated by viral N protein. The relevance of this interaction in virus life cycle was studied using small molecule drugs that disrupt host cytoskeleton and lipid raft. The results obtained suggested that the host cytoskeleton, especially actin-filaments facilitate virus release from the plasma membrane. RPV matrix protein acquired detergent resistance in infected cells as well as in transfected cells. The pattern of detergent resistance suggested an association with the cytoskeleton or cytoskeleton associated proteins. However, results obtained from co-localisation studies in the presence of actin inhibitor and cold-ionic detergents are not consistent with the above observation. This property could be due to self-association of matrix protein.
URI: http://hdl.handle.net/2005/382
Appears in Collections:Microbiology and Cell Biology (mcbl)

Files in This Item:

File Description SizeFormat
G20920.pdf4.76 MBAdobe PDFView/Open

Items in etd@IISc are protected by copyright, with all rights reserved, unless otherwise indicated.

 

etd@IISc is a joint service of NCSI & IISc Library ||
Feedback
|| Powered by DSpace || Compliant to OAI-PMH V 2.0 and ETD-MS V 1.01