Dr. M. S. Shaila
Professor
Ph.D. (Indian Institute of Science, Bangalore. India)
Room No. 301
Phone: 2293 2702; 2360 0139
E-mail: shaila@mcbl.iisc.ernet.in
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Our research programme is concerned with (a) the molecular analysis of virus genes and virus proteins involved in transcription and replication of the genome, (b) analysis of immune responses to virus proteins, and (c) analysis of functional domains responsible for hemagglutinin-neuraminidase activities of HN protein and fusion activity of F protein of PPRV. Two morbilliviruses, Rinderpest virus (RPV) and Peste des petits ruminants virus (PPRV) have been intensely investigated under the above areas.
 

The molecular mechanisms governing the negative sense RNA genome transcription and replication are largely unknown in the case of paramyxoviruses and morbilliviruses. The genomes of negative strand RNA viruses are tightly encapsidated by the nucleocapsid protein N and this N-RNA complex serves as the template for viral transcription and replication. Within the virion, the RNA dependent RNA polymerase (L protein) is associated with the N-RNA template through its interaction with the transcription factor, phosphoprotein P, to form the transcribing ribonucleoprotein (RNP) complex. L protein is the catalytic subunit of the polymerase complex and exhibits a number of enzymatic activities including the transcriptase and replicase activities. The product mRNAs are monocistronic and unencapsidated. Replication (full length genomic RNA synthesis) is linked to the simultaneous encapsidation of the nascent RNA by the N protein. The individual functions of the polymerase/replicase subunits are not well defined. We have endeavoured to characterize the transcription/replication apparatus of Rinderpest (RPV) and Peste des petits ruminants virus (PPRV).

 

As a first step, we characterized the in vitro transcriptional activity of the nucleocapsids of Rinderpest virus isolated from purified virus or from infected cells. We have analyzed the functional domains of the RPV RNA polymerase complex, the interactions of the P & L, P & N as well as P with itself, employing the yeast two hybrid system. A strikingly new observation is that oligomerization of P is a prerequisite for its interaction with N. Further, we have mapped the domain on P involved in P oligomerization and the domains involved in its interaction with N. Three serine residues in the amino terminal region, S49, S88 and S151 are involved in the phosphorylation of P by CKII. A phosphorylation-null mutant containing serine to alanine mutations in all the three residues is inactive in in vivo replication and transcription of Rinderpest virus minigenome. However, it plays a hitherto unidentified role in bringing about increased replication of the virus as well as increased in vivo replication/transcription of synthetic minigenome. The domains on L interacting with P and N proteins have also been mapped. The role of QGDNQ motif present in L protein has been assessed using site directed mutants of L in minigenome transcription-replication assay as well as in vitro reconstitution system.

 

The second aspect of work being focussed relates to the nature of immune response elicited in target animals by recombinant proteins expressed as extracellular virus (ECV). The helper T cell response in cattle has been mapped to two domains on RPV H protein. The CTL responses to ECV immunization have been studied and a CTL domain has been identified on RPV H protein. In collaboration with Prof. G. Lakshmi Sita, we have also generated transgenic peanut and pigeon pea plants expressing the hemagglutinin protein of Rinderpest virus and hemagglutinin-neuraminidase protein of PPRV. Immune responses to plant derived antigens in the target animals are elicited upon oral feeding in the absence of mucosal adjuvant.

 

The third area of work focusses on the identification of functional domains of viral proteins and solving the three dimensional structure of functional domains by X-Ray crystallography, in collaboration with Prof. N. Shamala of Physics Department. A structure-function relationship study on RPV P protein has provided important information on the involvement of P in viral transcription and replication. P protein exists as a tetramer, independent of its phosphorylation status. The tetramerization through four-stranded coiled structures is essential for function and remains conserved between RPV and PPRV.

 

The F protein of PPRV exhibits fusion activity without the fusion promotion function, a property unique for any morbillivirus. Therefore, we have used this biological activity of F protein of PPRV to study the role of the heptad repeats (HR1 and HR2) present on the F1 subunit in fusion event. HR1 and HR2 peptides interact with each other, forming a stable complex which is more structured. A three dimensional model has been generated for this fusion core complex, according to which the HR1-HR2 complex is a six helix coiled coil bundle and these two peptides inhibit virus-mediated fusion process.

 
 

SELECTED PUBLICATIONS

Sinnathamby, G., Renukaradhya, G. J., Rajasekhar, M., Nayak, R. and Shaila, M. S. (2001). Recombinant hemagglutinin protein of Rinderpest virus expressed in insect cell induces cytotoxic T cell responses in cattle. Viral Immunol. 14, 349-358. publications\ARTICLE.PDF.

Seth, S. and Shaila, M. S. (2001) The hemagglutinin-neuraminidase protein of Peste des petits ruminants virus is biologically active when transiently expressed in mammalian cells. Virus Res. 75, 169-177. publications\shaguna.pdf.

Seth, S. and Shaila, M. S. (2001) The fusion protein of Peste des petits Ruminants virus mediates biological fusion in the absence of hemagglutinin-neuraminidase protein. Virology 289, 86-94. publications\Virol_shaguna.pdf.

Renukaradhya, G. J., Mitra-Kaushik, S., Sinnathamby, G., Rajasekhar, M. and Shaila, M. S. (2002) Mapping of B cell epitopes of Hemagglutinin protein of Rinderpest virus. Virology 298, 214-223. publications\viro2.pdf

Renukaradhya, G. J., Sinnathamby, G., Seth, S., Rajasekhar, M. and Shaila, M. S. (2002) Mapping of B cell epitopic sites and identification of functional domains on the hemagglutini-neuraminidase protein of Peste des petits ruminants virus. Virus Res. 90, 171-185. publications\virology.pdf

Rahaman, A., Srinivasan, N., Shamala, N. and Shaila, M. S. (2003) The fusion core complex of the Peste des petits ruminants is a six-helix bundle assembly. Biochemistry 42, 922-931. publications\biochemistry.pdf

Renukaradhya, G.J., Suresh, K.B., Rajasekhar, M. and Shaila, M.S. (2003). Competitive enzyme-linked immunosorbent assay based on monoclonal antibody and recombinant Hemagglutinin for serosurveillance of Rinderpest virus. Journal of Clinical Microbiology, 41(3), 943-947. publications\JCMPaper.pdf

Abha Khandelwal, Lakshmi Sita, G. and Shaila, M.S. (2003). Expression of Hemagglutinin protein rinderpest virus is transgenic tobacco and immunogenicity of plant-derived protein in a mouse model. Virology, 308, 207-215. publications\virology'03.pdf

S. Basak, Tamal Raha, D. Chattopadhyay, A. Mazumdar, M.S. Shaila and D.J. Chattopadhyay (2003). Leader RNA binding ability of Chandipura virus P protein is regulated by its phosphorylation status: a possible role in genome transcription-replication switch. Virology, 307, 372-385. publications\virology_tamal.pdf

Raj, G.D., Kumar, A.S.S., Shaila, M.S., Nachimathu, K., Palaniswami, K.S. (2003). Molecular epidemiology of peste des petits ruminants viruses from Southern India. Veterinary Record, 152(9), 264-266.

Satyavathi, V.V., Prasad, V., Abha Khandelwal, Shaila, M.S. and Lakshmi Sita, G. (2002). Expression of hemagglutinin protein of Rinderpest virus in transgenic pigeon pea [Cajanus cajan (L) Millsp.] plants. Plant Cell Reports (in press).

Abha Khandelwal, G. Lakshmi Sita and M.S. Shaila (2003). Oral immunization of cattle with hemagglutinin protein or rinderpest virus expressed in transgenic peanut induces specific immune responses vaccine. 21: 3282-3289. publications\vaccine.pdf

Abha Khandelwal, K.J.M. Vally, N. Geetha, P. Venkatachalam, M.S. Shaila and G. Lakshmi Sita. Engineering hemagglutinin (H) gene of Rinderpest virus into peanut (Arachis hypogea L) for a possible source of vaccines. Plant Science 165: 77-84.

Prasad, V., Satyavathi, V.V., Sanjaya, Valli, K.M., Abha Khandelwal, Shaila, M.S. and Lakshmi Sita G. (2003). Expression of biologically active Hemagglutinin-neuraminidase protein of Peste des petits ruminants virus in transgenic pigeon pea [Cajanus cajan (L) Millop.]. Plant Science doi 10.1016/j.plantsci.2003.08.020.

Raha, T., Anasuya, C. and Shaila, M.S. (2004). Development of a reconstitution system for Rinderpest virus RNA synthesis in vitro. Virus Research 99: 131-138.publications\virusres04-01

Anasuya C., Raha, T. and Shaila, M.S. (2004). Effect of single amino acid mutations in the conserved GDNQ motif of L protein of Rinderpest virus on RNA synthesis in vitro and in vivo. Virus Research 99: 139-145. publications\virusres04-02

 

 


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