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.
| Identifer | oai:union.ndltd.org:IISc/oai:etd.ncsi.iisc.ernet.in:2005/382 |
| Date | 08 1900 |
| Creators | Subhashri, R |
| Contributors | Shaila, M S |
| Source Sets | India Institute of Science |
| Language | en_US |
| Detected Language | English |
| Type | Thesis |
| Rights | I grant Indian Institute of Science the right to archive and to make available my thesis or dissertation in whole or in part in all forms of media, now hereafter known. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. |
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