Asit K. Pattnaik, Ph.D.

Nebraska Center for Virology & Department of Veterinary and Biomedical Sciences University of Nebraska-Lincoln E126 Beadle Center, 1901 Vine Street, Lincoln, NE 68588-0666. Telephone: (402) 472-1067 Fax: (402) 472-8722 E-mail: apattnaik2@unl.edu

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 PUBLICATIONS:

1. Li, T. and Pattnaik, A.K. (1997). Replication signals in the genome of vesicular stomatitis virus and its defective interfering particles: identification of a sequence element that enhances DI RNA replication. Virology 232:248-259.

2. Pattnaik, A.K., Hwang, L., Li, T., Englund, N., Mathur, M., Das, T., and Banerjee, A.K. (1997). Phosphorylation within the amino-terminal acidic domain I of the phosphoprotein of vesicular stomatitis virus is required for transcription but not for replication. J. Virology 71:8167-8175.

3. Das, T., Pattnaik, A.K., Takacs, A., Li, T., Hwang, L., and Banerjee, A.K. (1997). Basic amino acid residues at the carboxy-terminal eleven amino acid region of the phosphoprotein (P) are required for transcription but not for replication of vesicular stomatitis virus genome RNA. Virology, 238:103-114.

4. Hwang, L.N., Englund, N., and Pattnaik, A.K. (1998). Polyadenylation of vesicular stomatitis virus mRNA dictates efficient transcription termination at the intergenic gene junctions. J. Virology, 72:1805-1813.

5. Li, T. and Pattnaik, A.K. (1999). Overlapping signals for transcription and replication at the 3'-terminus of vesicular stomatitis virus genome. J. Virology, 73:444-452.

6. Hwang, L.N., Englund, N., Das, T., Banerjee, A.K., and Pattnaik, A.K. (1999). Optimal replication activity of VSV RNA polymerase requires phosphorylation of residues at the carboxy-terminal domain II of its accessory subunit, the phosphoprotein, P. J. Virology 73:5613-5620.

7. Balachandran, S., Roberts, P.C., Brown, L.E., Truong, H., Pattnaik, A. K., Archer, D.R., and Barber, G.N. (2000). Essential role for the dsRNA-dependent protein kinase, PKR, in innate immunity to viral infection. Immunity, 13:129-141.

8. Das, S. C. and Pattnaik, A. K. (2004). Phosphorylation of vesicular stomatitis virus phosphoprotein P is indispensable for virus growth. J. Virology 78:6420-30.

9. Truong, H. M., Lu, Z., Kutish, G., Galeota, J., Osorio, F. A., and Pattnaik, A.K. (2004). 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.

10. Zhang, J., Das, S. C., Kotalik, 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. J Biol Chem. 279(44): 46335-46342. [Epub ahead of print, Aug. 20, 2004]

11. Das S. C. and Pattnaik, A. K. (2005). Role of the hypervariable hinge region of vesicular stomatitis virus in viral RNA synthesis and assembly of infectious virus particles. J. Virology, (In press).

12. Fang, Y., Faaberg, K. S., Rowland, R., Christopher-Hennings, J., Pattnaik, A. K., Osorio, F. A., and Nelson, E. A. (2006). Construction of a full-length cDNA infectious clone of a European-like Type 1 PRRSV isolated in the U.S. In: The Nidoviruses: The Control of SARS and Other Nidovirus Diseases. edited by S. Perlman and K. Holmes, pp. 605-608.

13. Liu, S., Ansari, I. H., Das, S. C., and Pattnaik, A. K. (2006). 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.

14. Ansari, I. H., Kwon, B. J., Osorio, F. A., and Pattnaik, A. K. (2006). Influence of N-Linked Glycosylation of Porcine Reproducitive and Respiratory Syndrome Virus GP5 on Virus Infectivity, Antigenicity and Ability to Induce Neutralizing Antibodies. J. Virology, 80(8): 3994-4004.

15. Das, S. C., Nayak, D., Zhou, Y., and Pattnaik, A. K. (2006). Visualization of Intracellular Transport of Vesicular Stomatitis Virus Nucleocapsids in Living Cells. J. Virology, (In Press).