Asit K. Pattnaik, Ph.D.
Research Program: My laboratory has been involved in studies of the mechanisms and regulation of transcription and replication of RNA virus genomes. As models, we have been using vesicular stomatitis virus (VSV), a non-segmented negative-strand RNA virus in the order mononagavirales and porcine reproductive and respiratory syndrome virus (PRRSV), a positive-strand RNA virus in the order nidovirales. Understanding the mechanism of gene expression and its regulation is essential to identifying unique virus-specific targets for therapeutic intervention in controlling infection. We developed a complete cDNA-based reverse genetic system for VSV that allows us to introduce specific genetic alterations into the genome of VSV and analyze the effects of these alterations on transcription and replication of the viral genome. This system laid the foundation for genetic manipulation of negative-strand RNA viruses and has been used extensively by investigators worldwide to generate infectious molecular clones of almost all the negative-strand RNA viruses. Using the system, three major areas of research are currently being investigated in my laboratory: (1) to understand the role of nucleotide sequences within the viral genome that control transcription and replication processes, (2) to understand structure-function relationships of the viral proteins involved in genome replication and transcription, and (3) to generate and characterize recombinant VSV encoding heterologous viral proteins for vaccine development. Our studies have revealed that specific sequences present at the termini as well as at the intercistronic junctions of VSV genome are required for transcription of individual virus mRNA. Sequences at the termini are also required for efficient replication of the viral genome. In addition, we 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. These studies have led to a model suggesting that transcriptase and replicase of VSV are two distinct molecular complexes. Further studies are focused on characterizing these complexes and examining their structure. We have recently determined the nucleotide sequence of the approximately 15.5 kilobase RNA genome of PRRSV. A cDNA representing the entire genome was assembled. RNA transcribed in vitro from the plasmid encoding the genome was shown to produce infectious PRRSV when transfected into cells. Using this infectious cDNA clone, we are addressing questions regarding replication and pathogenesis of this important swine pathogen. Further studies to examine the role of sequences within the viral genome that control transcription and replication and also the role of viral proteins in these processes are being carried out. Selected Recent Publications: 1. Otsuka, M., Jing, Q., Georgel, P., New, L., Chen, J., Mols, J., Kang, Y. J., Jiang, Z., Du, X., Cook, R., Das, S. C., Pattnaik, A. K., Beutler, B., and Han, J. Hypersusceptibility to vesicular stomatitis virus infection in dicer1-deficient mice is due to impaired miR24 and miR93 expression. Immunity, 27: 123-134, 2007. 2. Kwon, B. J., Ansari, I. H., Osorio, F. A., and Pattnaik, A. K. Infectious clone-derived viruses from virulent and vaccine strains of porcine reproductive and respiratory syndrome virus mimic biological properties of their parental viruses in a pregnant sow model. Vaccine, 24(49-50): 7071-80, 2006. 3. de Lima, M., Pattnaik, A. K., Flores, E. F., and Osorio, F. A. Serologic marker candidates identified amongst B-cell linear epitopes of Nsp2 and structural proteins of a North American strain of porcine reproductive and respiratory syndrome virus. Virology, 353: 410-421, 2006. 4. Das, S. C., Nayak, D., Zhou, Y., and Pattnaik, A. K. Visualization of intracellular transport of vesicular stomatitis virus nucleocapsids in living cells. J. Virology, 80: 6368-6377, 2006. 5. Ansari, I. H., Kwon, B. J., Osorio, F. A., and Pattnaik, A. K. Influence of N-linked glycosylation of porcine reproductive and respiratory syndrome virus GP5 on virus infectivity, antigenicity, and ability to induce neutralizing antibodies. J. Virology, 80: 3994-4004, 2006 6. Liu, S., Ansari, I. H., Das, S. C., and Pattnaik, A. K. Insertion and deletion analyses identify regions of nonstructural protein 5A of hepatitis C virus that are dispensable for viral genome replication. J. General Virology, 87: 323-327, 2006. 7. Das, S. C. and Pattnaik, A. K. Role of the hypervariable hinge region of the phosphoprotein P of vesicular stomatitis virus in viral RNA synthesis and assembly of infectious virus particles. J. Virology, 79: 8101-8112, 2005. 8. Zhang, J., Das, S. C., Kotalik, C., Pattnaik, A. K., and Zhang, L. The latent membrane protein 1 of Epstein-Barr virus establishes an antiviral state via induction of interferon-stimulated genes. J. Biol. Chem., 279(44): 46335-46342, 2004. 9. Truong, H. M., Lu, Z., Kutish, G., Galeota, J., Osorio, F. A., and Pattnaik, A. K. A highly pathogenic porcine reproductive and respiratory syndrome virus generated from an infectious cDNA clone retains the in vivo virulence and transmissibility properties of the parental virus. Virology, 325: 308-19, 2004. 10. Das, S. C. and Pattnaik, A. K. Phosphorylation of vesicular stomatitis virus phosphoprotein P is indispensable for virus growth. J. Virology, 78: 6420-30, 2004. |
|
