Subash C. Das, DVM, MVS, PhD.

Research Assistant Professor Veterinary and Biomedical Sciences University of Nebraska-Lincoln 1901 Vine Street, E131 Beadle Center, Lincoln, NE-68588 Ph: 402-472-1166 Fax: 402-472-8722 E-mail: sdas2@unlnotes.unl.edu

Research Interests

The two viruses I am studying are vesicular stomatitis virus (VSV), a non-segmented negative-strand RNA virus and porcine reproductive and respiratory syndrome virus (PRRSV), a non-segmented positive-strand RNA virus. Due to its simple genome organization VSV has served as an attractive model to study the gene expression in negative-stranded RNA viruses. Understanding the mechanism of gene expression and its regulation is essential to identifying unique virus-specific targets for therapeutic intervention in controlling infection. More specifically I am looking at the role of VSV phosphoprotein P in viral transcription, replication and assembly of infectious virus particles. Phosphoprotein of VSV is a multifunctional protein which is an essential subunit of viral polymerase. Using reverse genetics I have demonstrated that phosphorylation at specific residues within the P protein of VSV regulates the activities of the viral RNA-dependent RNA polymerase in transcription and replication and plays a major role in the life cycle of VSV. Using transposon-insertion and deletion mutagenesis we recently found out that the hypervariable hinge region of VSV P protein plays an important role in viral RNA synthesis and assembly of infectious particles. At present we are mapping out the individual amino acids in the hypervariable region of P that is required for virus assembly. Currently efforts are being made to establish a yeast-two-hybrid system to identify the cellular /viral factors involved in the assembly of VSV. We are further planning to investigate the role of nucleotide sequences within the viral genome that control encapsidation, transcription and replication processes.

We have made use of our recent finding that the hypervariable region of VSV P protein can tolerate insertion of 19 amino acids with minimal effect on P protein activity. This has led us to produce a fluorescently labeled VSV with the eGFP inserted at the hypervariable region of P protein. Using this green virus we are investigating the transport of viral nucleocapsids by time lapse microscopy. This has allowed us to track the movement of individual nucleocapsids in infected cells. We have demonstrated that microtubules play an important role in the transport of VSV nucleocapsids from the site of synthesis to the site of assembly and mitochondria may play a role in this process. Several leads in this direction include single-particle tracking of viral nucleocapsids, multicolor live-cell imaging of ribonucleoprotein complexes and identification of microtubule motors involved in the transport.

Another aspect of my work has been the development of viral vaccines by genetic manipulations. At present I am using VSV as a vector to express porcine respiratory and reproductive syndrome virus (PRRSV) glycoproteins to study the immunogenicity of these proteins in animals. Recombinant VSVs expressing PRRSV GP5 and M proteins have been recovered by reverse genetics. Using these recombinant viruses we further plan to study the mechanism of entry and tissue tropism in PRRSV infection. Animal experiments are also being carried out for testing these recombinant viruses for generation of humoral and cell-mediated immune responses against PRRSV and to explore the possibility of using them as vaccines for the prevention of PPRSV infection.

Selected Publications

1. Liu, S., Ansari, IH., Das S.C. and Pattnaik, A.K. (2005) Insertion and deletion analyses identify regions of nonstructural protein 5A of hepatitis C virus that are dispensable for viral genome replication. Journal of Virology. In Press.

2. Das, S.C. and Pattnaik, A. K. (2005) Role of the hypervariable hinge region of the phosphoprotein P of vesicular stomatitis virus in viral RNA synthesis and assembly of infectious virus particles. Journal of Virology. 79: 8101-8112.

3. Zhang, J., Das, S.C., Kotalika, C., Pattnaik, A.K. and Zhang, L. (2004) The latent membrane protein 1 of Epstein-Barr virus establishes an antiviral state via induction of interferon-stimulated genes. Journal of Biological Chemistry. 279: 46335-46342.

4. Das, S.C. and Pattnaik, A.K. (2004). Phosphorylation of residues within the amino- and carboxy-terminal domains of the phosphoprotein P of vesicular stomatitis virus is indispensable for virus growth. Journal of Virology.78: 6420-6430.

5. Barrett, T., Parida, S., Mohapatra, M., Walsh, P., Das, S., and Baron, M.D. (2003). Development of new generation Rinderpest vaccines. Developments in Biologicals. 114: 89-97. Review. Brown F, Roth J (eds): Vaccines for OIE List A and Emerging Animal Diseases. Dev Biol. Basel, Karger, 2003, vol 114, pp 89-97.

6. Das, S.C., Baron M.D. and Barrett, T. (2000). Recovery and characterisation of a chimeric rinderpest virus with the glycoproteins of peste des petits ruminants virus: homologous F and H proteins are required for virus viability. Journal of Virology. 74: 9039-9047.

7. Das, S.C., Baron, M.D., Skinner, M.A. and Barrett, T. (2000). Improved technique for transient expression and negative strand virus rescue using fowl pox T7 recombinant virus in mammalian cells. Journal of Virological Methods. 89: 119-127.