The long-term goal of this laboratory is to understand the molecular basis of pathogenicity of viruses belonging to the negative strand RNA (nsRNA) virus family using vesicular stomatitis virus (VSV) and human parainfluenza virus type 3 (HPIV-3) as the prototype viruses. The viruses, such as rabies, measles, mumps, parainfluenza, respiratory syncytial, and many more fall in this nsRNA virus category. A thorough understanding of the mode of transcription and replication of these viruses is fundamental to develop reagents to combat these deadly pathogens. Our major emphasis towards this goal has been to establish the functions of the key viral proteins, such as L, the RNA polymerase, P, the transcription factor and N, the nucleocapsid protein, encapsidating the genome RNA. The virus ribonucleoprotein (RNP) complex containing these polypeptides transcribes the genome RNA in vitro as well as in vivo by which it initiates infection within the infected cells. We also study the important area of host-virus interaction and discovered several host proteins that play critical roles in the gene expression of these viruses. Following is the summary of our current activities in VSV and HPIV-3 research:
Vesicular stomatitis virus:We have been successful in expressing in biologically active form, viral polypeptides that constitute the transcribing RNP, i.e., L,N, P proteins in procaryotic and eukaryotic cells using recombinant expression vectors. Several important discoveries were made especially with respect to the subunit composition of the L protein and the putative replicase complex. We have shown that L protein expressed in insect cells associates with the cellular translation elongation factor EF-1 abg subunits for its activity.
In this respect VSV RNA polymerase has a striking similarity with bacteriophage Qb replicase which requires the bacterial homologue of the translation elongation factors Ts Tu. This unique finding has the potential to unravel the roles of these cellular proteins in VSV RNA polymerase function. Using a reverse-genetics system to study transcription and replication of specially constructed minigenome or defective interfering (DI) particles using cDNAs encoding P proteins and wt L and N proteins, we have demonstrated that while the transcription complex is composed of L-P 2-3, the replicase appears to be a tripartite complex between L-(N-P) which initiates the replication reaction. Understanding the structure and function of the replicase complex is fundamental to gain insight into the replicative pathways in VSV life cycle.
Human parainfluenza virus: The transcription complex of HPIV-3 consists of L,P, and N proteins in which the N protein encapsidates the genome RNA. We demonstrated that specific interaction and polymerization of actin on the ribonucleoprotein (RNP) complex leads to the activation of transcription in vitro. We have further shown that the virus replicates in the cytoskeletal network where actin plays a critical role in the replication process. Continued studies along this line led to discovery of two additional cellular proteins that specifically interact with cis-acting viral RNAs. These include the key glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and the nuclear autoantigen, La protein. The involvement of GAPDH in HPIV-3 life cycle is particularly interesting, because it is a cellular metabolic enzyme, and its association with an RNA virus has not been previously reported. Understanding, in detail, the molecular basis of the interplay of viruses and cellular proteins would further our knowledge in understanding the role of the host in promoting virus replication, thus, providing opportunities to develop agents that specifically target these host-virus interactions.
Recently, a full-length cDNA clone of human parainfluenza virus type 3 (HPIV-3) genome (called pHPIV-3) was constructed, and recombinant, infectious HPIV-3 was generated by transfecting pHPIV-3 and support plasmids encoding the HPIV-3 Np, P, and L proteins into HeLa cells infected with a recombinant vaccinia virus which expresses T7 RNA polymerase. Recovered virus was neutralized by anti-HPIV-3 antisera and shown to contain specific base substitutions characteristic of pHPIV-3. Availability of an infectious clone for HPIV-3 will enhance our understanding of the molecular biology of the gene expression of HPIV-3. Moreover, the ability to introduce specific mutations in the infectious cDNA will help us to develop effective vaccine against this important human pathogen.
Bose, S., Mathur, M., Bates, P. Joshi, N., and Banerjee, A.K. Requirement for cyclophilin A for the replication of vesicular stomatitis virus New Jersey serotype. J. Gen. Virol. 84:1687-1699 (2003). [PubMed]
Gupta A.K., Shaji D. and Banerjee A.K. Identification of a novel tripartite complex involved in replication of vescicular stomatitis virus genome RNA. J Virol. 77:732-8 (2003). [PubMed]
Mathur M., and Banerjee, A.K. Novel Binding of GTP to the Phosphoprotein P of Vesicular Stomatitis Virus Type 3 (HPIV3). Gene Expression. 10:193-200 (2002). [PubMed]
Bose, S. and Banerjee, A.K. Role of Heparan Sulfate in Human Parainfluenza Virus Type 3 Infection. Virology 298:73-83 (2002). [PubMed]
Gao, J., De, B.P., and Banerjee, A.K. Interferon Type I Down-regulates Human Parainfluenza Virus Type-3 Induced Major Histocompatibility Complex Class II. J. Virol Immunology 15:85-93 (2002). [PubMed]