Dr. Padmanabhan is Professor of Microbiology & Immunology

Summary:

• Ph.D. Wayne State University, Detroit, MI., 1968
• At Georgetown Since: 2002
• Current lab has capacity for new Ph.D. students
• Contact: (202) 687-2092, rp55@georgetown.edu

Details:

The main focus of my laboratory has been to understand the mechanisms of flaviviral RNA replication and polyprotein processing in the dengue viral life cycle. Dengue viruses (types 1-4), members of Flaviviridae, are transmitted by mosquito vectors, Aedes aegypti and Aedes albopictus. They are recognized as the causative agents of diseases from dengue fever, a simple self-limiting disease, to more severe forms, dengue hemorrhagic fever/dengue shock syndrome, affecting about 100 million people annually worldwide with thousands of fatalities. Over 40% of the world population is at risk for dengue viral infections and currently there is no effective vaccine or antiviral drug available. Moreover, infections caused by another member of the mosquito-borne Flaviviridae, West Nile virus, have recently been rapidly expanding within the United States.

Additional Research Information:

Dengue viruses have positive-strand RNA genomes with a 5'-cap structure. The viral RNA codes for a single polyprotein precursor, which is processed by the host protease(s) as well as the two-component viral serine protease into at least 10 mature viral proteins. Processing by the viral protease requires the viral protein, NS2B, which acts as a cofactor to activate the N-terminal serine protease domain of NS3.

The mechanism and the amino acid residues involved in activation are unknown and are currently under study. An in vitro protease assay has been developed in our laboratory using E. coli-expressed and purified components of the viral protease. Using a purified protease, a high throughput screen with ~32,000 compounds was carried out for identication of small molecule inhitors. We have also developed a cell-based assay system using Dengue virus type 2 replicon to study the effects of nucleoside and non-nucleoside analogs on viral replication. The crystal structure of NS3 protease domain in the absence of NS2B was solved in collaboration with Dr. Krishna Murthy (Univ. of Alabama).

Recently, crystal structures of the proteases of dengue virus and West Nile virus were solved by the Novartis group (Erbel et al. Nat Struct Mol Biol. 2006 13(4):372-373). Using our in vitro protease assay in a high throughput form, we screened ~32,000 compounds from seven chemical libraries. Further analysis revealed a novel class of potential small molecule inhibitors of West Nile virus protease. The C-terminal region of NS3 has an RNA-stimulated NTPase/RNA helicase and 5'-RNA triphosphatase activities. In flavivirus-infected cells, NS3 and NS5, the RNA-dependent RNA polymerase (RdRP), exist as a complex.

An in vitro replication system has been developed that utilizes lysates from dengue virus-infected mosquito and mammalian cells or purified recombinant NS5 and exogenous viral template RNAs. The identification of proteins and RNA structural motifs involved in viral replication using the in vitro and a cell-based assay system are pursued in our laboratory.

Publications:

1. You, S. Falgout, B., Markoff, L, and Padmanabhan, R.: The in vitro replication system for dengue virus reveals that the conserved 3?-stem-loop structure is required for RNA synthesis by viral replicase. J. Biol. Chem. 276, 15581-15591, 2001.

2. Ackermann, M. and Padmanabhan, R..: De novo synthesis of RNA by the dengue virus RNA dependent RNA polymerase exhibits temperature dependence at the initiation but not elongation phase. J. Biol. Chem. 276, 39926-39937,2001.

3. Bartelma, G. and Padmanabhan, R. Expression, purification, characterization of 5?-RNA triphosphatase activity of dengue virus type 2. Virology, 2002 299, 122-132, 2002.

4. Nomaguchi M., Ackermann M, Yon C., You S, and Padmanabhan, R. De Novo Synthesis of Negative-Strand RNA by Dengue Virus RNA-Dependent RNA Polymerase In Vitro: Nucleotide, Primer, and Template Parameters. J. Virology 77, 8831-8842, 2003.

5. Nomaguchi1, M, Teramoto, T., Yu, L., Markoff, L, Padmanabhan1, R. Requirements for West Nile Virus Minus- and Plus-Strand Subgenomic RNA Synthesis in vitro by the viral RNAdependent RNA Polymerase Expressed in E. coli. J. Biol. Chem. 279:12141-51, 2004.

6. Ganesh, V.K., Mueller, N., Judge, K., Luan, C.-H., Padmanabhan, R., and Murthy, K.H.M. Identi.cation and characterization of nonsubstrate based inhibitors of the essential dengue and West Nile virus proteases. Biorg. and Med Chem. 13, 257-264, 2005.

7. Yon, C., Teramoto, T., Mueller, N., Phelan, J., Ganesh, V. K., Murthy, K. H. Padmanabhan, R. Modulation of the nucleoside triphosphatase/RNA helicase and 5'-RNA triphosphatase activities of dengue virus type 2 nonstructural protein 3 (NS3) by interaction with NS5, the RNA-dependent RNA polymerase. J. Biol. Chem. 280, 27412-19, 2005.

8. Takhampunya, R., Ubol, S., Houng, H-S., Cameron, C.E., and Padmanabhan, R. Inhibition of dengue virus replication by mycophenolic acid and ribavirin. J. Gen. Virol. 87, 1947-52, 2006.

9. Takhampunya, R., Padmanabhan, R., and Ubol, S. Antiviral action of nitric oxide on dengue virus type 2 replication. J. Gen. Virol. 87, 2006 (in press).

10. Yocupicio-Monroy, M., Padmanabhan, R., Medina, F., and del Angel, R. M. Mosquito La protein binds to the 3? Untranslated Region of the Positive and Negative Polarity Dengue virus RNAs and Relocates to the Cytoplasm of Infected Cells Virology 2006 (in press).