Asit K. Pattnaik, Ph.D.

Professor University of Nebraska-Lincoln School of Veterinary and Biomedical Sciences 109 Morrison Center Lincoln, NE 68583-0900 Telephone: (402) 472-1067 Fax: (402) 472-3323 E-mail: apattnaik2@unl.edu

Research Program:

            Studies in my laboratory focus on molecular biology and immunopathogenesis of two different viral pathogens, the vesicular stomatitis virus (VSV), a non-segmented negative-strand RNA virus in the family Rhabdoviridae and order Mononagavirales, and the porcine reproductive and respiratory syndrome virus (PRRSV), a positive-strand RNA virus in the family Arteriviridae and order Nidovirales. VSV has served as an excellent paradigm for many negative-strand RNA viruses (some of which are important human pathogens, such as respiratory syncytial, rabies, measles, and parainfluenza, hemorrhagic bunya and arenaviruses) to understand the basic mechanisms of the genetic expression of this group of viruses. PRRSV is an economically important pathogen, causing serious diseases in swine worldwide. Understanding the mechanism(s) of gene expression and its regulation as well as pathogenesis is essential for identifying virus-specific targets for therapeutic intervention in controlling infection by these viruses.
            Using VSV, we had previously developed a completely cDNA-based reverse genetic system (Cell, 69:1011-1020, 1992), which has allowed us to introduce specific genetic alterations into the genome of VSV and its defective interfering particles and examine the effects of these alterations on transcription and replication of the viral genomes. The system laid the foundation for genetic manipulation of all negative-strand RNA viruses and has been used extensively by investigators worldwide to generate infectious molecular clones of the negative-strand RNA viruses. Using the system, previous studies from our laboratory addressed three major areas of research: (1) understand the role of nucleotide sequences within the viral genome that control transcription and replication processes; (2) understand structure-function relationships of the viral proteins involved in genome replication, transcription, and virus assembly; and (3) generate and characterize recombinant VSV encoding heterologous viral proteins for vaccine development.
            In recent studies, we have described recombinant VSV with a fluorescently tagged P protein (PeGFP fusion protein) to examine intracellular transport of viral nucleocapsids from the sites of synthesis to sites of virus assembly (JVI, 80:6368-6377, 2006). Furthermore, we have generated dual-fluorescent viruses, which have been used to examine by live cell imaging, the entry and uncoating as well as assembly of the viral nucleocapsids in infected cells (JVI, 83:2611-2622, 2009). To understand the regulation of viral genome transcription and replication, we employed structure-guided mutational studies on the viral nucleocapsid protein and have shown (JVI, 83:5525-5534, 2009) that the nucleocapsid protein structure and the resulting nucleocapsid (N-RNA) template are key determinants of N-RNA template functions (transcription and replication). Further studies are being carried out to understand how the nucleocapsid protein and the viral polymerase interactions modulate the transcription and replication functions of the viral template.
            To understand the role of cellular functions in regulating viral replication, in collaboration with Dr. Han’s group at Scripps Research Institute at La Jolla, San Diego, we have recently demonstrated that mammalian dicer plays an integral role in innate antiviral immunity to VSV (Immunity, 27:123-134, 2007) through generation of specific microRNAs. We are currently examining the role of host cell functions in viral genome replication by employing genome-wide siRNA screens. A human genome-wide siRNA screening study completed recently for VSV (Proc. Natl. Acad. Sci., USA, In Press, 2011) has identified many important cellular pathways and host cell factors required for infection and replication of VSV. Most interestingly, these studies have identified coatomer protein complex I (COPI) being critically involved in gene expression of VSV as well as two other cytoplasmically replicating negative-strand RNA viruses, the lymphocytic choriomeningitis virus (LCMV) and the human parainfluenza virus type 3 (HPIV3). Further studies have also identified cellular poly(C) binding proteins as important regulators of VSV gene expression (JVI, 85: 9459-9471, 2011). Current studies are directed at understanding of the mechanisms of the involvement of these cellular proteins and pathways in VSV gene expression.      
            For studies related to PRRSV replication and immune response in infected pigs and cultured cells, we first the nucleotide sequence of the approximately 15.5 kilobase RNA genome and assembled a cDNA clone of the entire viral genome from which infectious virus could be readily recovered (Virology, 325:308-319, 2004). Using this infectious cDNA clone, we are addressing several questions regarding replication and pathogenesis of this important swine pathogen. We have shown that the PRRSV evades host cells’ immune response by employing “glycan shielding” mechanism (JVI, 80:3994-4004, 2006; JVI, 85:5555-5564, 2011), which will have significant impact on PRRSV vaccine development. We have also constructed an infectious clone of a vaccine strain of PRRSV (Vaccine, 24:7071-80, 2006) and by generating chimeric viruses between the highly pathogenic strain and the attenuated vaccine strain we have mapped the regions of the viral genome critical for virulence and attenuation of PRRSV (Virology, 380:371-378, 2008). Our studies have identified the viral glycoproteins that interact with the receptor for entry of the virus into the host cells (JVI, 84:1731-1740, 2010; Virology, 410:385-394, 2011). Studies are being conducted to block these interactions to reduce virus infections. We have recently shown that several of the PRRSV nonstructural proteins possess the inherent ability to suppress the host cell’s innate immune response (JVI, 84:1574-1584, 2010; Virology, 406: 270-279, 2010; JVI, 85(24), In Press, 2011). These studies along with other ongoing studies in our laboratory in collaboration with the laboratory of Dr. F. A. Osorio position us to generate second generation differential PRRSV vaccines by reverse genetic methods.

Selected Recent Publications:

Panda, D., Das, A., Dinh, P. X., Subramaniam, S., Nayak, D., Barrows, N. J., Pearson, J. L., Thompson, J., Kelly, D. L., Ladunga, I., and Pattnaik, A. K. (2011). RNAi Screening Reveals a Requirement for Host Cell Secretory Pathway in Infection by Diverse Families of Negative-Strand RNA Viruses. Proc. Natl. Acad. Sci., USA, In Press.

Beura, L. K., Dinh, P. X., Osorio, F. A., and Pattnaik, A. K. (2011). Cellular Ploy(C) Binding Proteins 1 and 2 Interact with Porcine Reproductive and Respiratory Syndrome Virus Non-Structural Protein 1b and Support Viral Replication. J. Virology, 85(24), Epub ahead of print, October 5. PMID: 21976648

Dinh, P. X., Beura, L. K., Panda, D., Das, A., and Pattnaik, A. K. (2011). Antagonistic Effects of Cellular Poly(C) Binding Proteins on Vesicular Stomatitis Virus Gene Expression. J. Virology, 85(18): 9459-9471. PMID: 21752917

Vu, H. L. X., Kwon, B. J., Yoon, K.-J, Laegreid, W. W., Pattnaik, A. K., and Osorio, F. A. (2011). Immune Evasion of porcine reproductive and respiratory syndrome virus through glycan shielding involved glycoprotein 5 as well as glycoprotein 3. J. Virology,85(11): 5555-5564. PMID: 21411530

Panda, D. and Pattnaik, A. K. (2011). Transcription of Vesicular Stomatitis Virus RNA Genome. In: Negative Strand RNA Virus (Edited by: Luo, M.), Chapter 8, pp.149-174, WorldScientific Publishing Co., Hackensack, New Jersey, USA.

Subramaniam, S., Sur, J.-H., Kwon, B. J., Pattnaik, A. K., and Osorio, F. A. (2011). A virulent strain of porcine reproductive and respiratory syndrome virus does not up-regulate interleulin-10 levels in vitro or in vivo. Virus Research, 155: 415-422. PMID: 21168453

Das, P.B., Vu, H.L., Dinh, P.X., Cooney, J.L., Kwon, B., Osorio, F.A., Pattnaik, A.K. (2011). Glycosylation of minor envelope glycoproteins of PRRSV in infectious virus recovery, receptor interaction, and immune response. Virology,410(2):385-394. PMID: 21195444

Subramaniam, S., Kwon, B. J., Beura, L. K., Kuszynski C. A., Pattnaik, A. K., and Osorio, F. A. (2010). Porcine reproductive and respiratory syndrome virus non-structural protein 1 suppresses tumor necrosis factor-alpha promoter activation by inhibiting NF-B and Sp1. Virology, 406: 270-279. PMID: 20701940

Pattnaik, A. K. and Panda, D. (2010). Biarsenical Labeling of Tetracysteine-Tagged Proteins. In Imaging: A Laboratory Manual, Edited by R. Yuste, Cold Spring Harbor Press, pp. 291-298, New York, NY.

Panda, D., Dinh, P. X., Beura, L. K., and Pattnaik, A. K. (2010). Induction of Interferon and Interferon Signaling Pathways by Replication of Defective Interfering Particle RNA in Cells Constitutively Expressing Vesicular Stomatitis Virus Replication Proteins. J. Virology, 84:4826-4831. (PMID: 20181705)

Das, P. B., Dinh, P. X., Ansari, I. H., de Lima, M., Osorio, F. A., and Pattnaik, A. K. (2010). The Minor Envelope Glycoproteins GP2a and GP4 of Porcine Reproductive and Respiratory Syndrome Virus Interact with the Receptor, CD163. J. Virology, 84: 1731-1740. PMID: 19939927

Beura, L. K., Sarkar, S. N., Kwon, B. J., Subramaniam, S., Jones, C., Pattnaik, A. K., and Osorio, F. A. (2010). Porcine Reproductive and Respiratory Syndrome Virus nonstructural protein nsp1 modulates host immune response by antagonizing IRF3 activation. J. Virology, 84: 1574-1584. PMID: 19923190

de Lima, M.  Ansari, I. H., Das, P. B., Ku, B., Martinez-Lobo, F. J., Pattnaik, A. K., and Osorio, F. A. (2009). GP3 is a Structural Component of the PRRSV Type II Virion. Virology, 390:31-36. PMID: 19467555

Nayak, D., Panda, D., Das, S. C., Luo, M., and Pattnaik, A. K. (2009). Single Amino Acid Alterations in a Highly Conserved Central Region of Vesicular Stomatitis Virus N Protein Differentially Affect the Viral Nucleocapsid Template Functions. J. Virology, 83:5525-5534. (PMID: 19321605)

Das, S. C., Panda, D., Nayak, D., and Pattnaik, A. K. (2009). Biarsenical Labeling of Vesicular Stomatitis Virus Encoding Tetracysteine-Tagged M Protein Allows Dynamic Imaging of M Protein and Virus Uncoating in Infected Cells. J. Virology, 83:2611-2622. (PMID: 19153240)

de Lima, M., Kwon, B., Ansari, I. H., Pattnaik, A. K., Flores, E. F. , and Osorio, F. A. (2008). Development of a porcine reproductive and respiratory syndrome virus differentiable (DIVA) strain through deletion of specific immunodominant epitopes. Vaccine, 26:3594-3600. PMID: 18538899

You, J. H., Howella, G., Pattnaik, A. K., Osorio, F. A., and Hiscox, J. A.  (2008). A model for the dynamic nuclear/nucleolar/cytoplasmic trafficking of the porcine reproductive and respiratory syndrome virus nucleocapsid protein based on live cell imaging. Virology, 378:34-47. PMID: 18550142

Kwon, B. J., Ansari, I. H., Pattnaik, A. K., and Osorio, F. A. (2008). Identification of virulence determinants of Porcine Reproductive and Respiratory Syndrome virus through construction of chimeric clones. Virology, 380:371-378. PMID: 18768197

Luyet, P. P. T. Falguieres, V. Pons, A. K. Pattnaik, and J. Gruenberg. (2008). The ESCRT-I Subunit TSG101 Controls Endosome-to-Cytosol Release of Viral RNA. Traffic, 9:2279-2290. (PMID: 18817529)

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. (2007). Hypersusceptibility to vesicular stomatitis virus infection in dicer-deficient mice is due to impaired miR24 and miR93 expression. Immunity, 27:123-134. (PMID: 17613256)

Kwon, B. J., Ansari, I. H., Osorio, F. A., and Pattnaik, A. K. (2006). 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. PMID: 17049689

de Lima, M., Pattnaik, A. K., Flores, E. F., and Osorio, F. A. (2006). 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. PMID: 16843516

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, 80:6368-6377. (PMID: 16775325)

Ansari, I. H., Kwon, B. J., Osorio, F. A., and Pattnaik A. K. (2006). 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. PMID: 16571816

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. (PMID: 16432018)

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. J. Virology, 79:8101-8112. (PMID: 15956555)

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. PMID: 15322136

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. PMID: 15246270

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. (PMID: 15163735)