Research Projects

Nipah virus is categorized as a biosafety level (BSL)-4 pathogen, and therefore, only organizations with BSL-4 facilities can work with it. Hence, I plan to develop a minigenome system that can be used in a BSL-2 facility (which is available in most Indian research institutes) using the genome sequence of the Nipah virus isolated in India. This will expedite research to understand the molecular mechanisms underlying Nipah virus transcription and replication. To the best of my knowledge, there is currently no Nipah virus minigenome generated using the genome sequence of an Indian Nipah virus isolate. The Nipah virus minigenome RNA will contain all the essential RNA sequences required for Nipah virus transcription and replication. However, it will lack sequences coding for viral proteins, instead expressing a reporter gene, such as luciferase or eGFP. Out of the six genes, only three viral genes expressing proteins—N, P, and L—essential for viral transcription and replication will be expressed using three separate plasmids. Hence, this minigenome system will be suitable for a BSL-2 facility. I plan to generate a cell line that stably expresses T7 polymerase tagged with an auxin-inducible degron (AID) tag to avoid the presence of viral genomic RNA synthesized by T7 polymerase in the later stage of the assay to study viral RNA synthesis exclusively by the viral polymerase. This approach is very novel. A minigenome system expressing a reporter gene, such as luciferase, will provide an assay system for high-throughput screening of anti-Nipah molecules. As the Nipah virus minigenome would be developed in an Indian research lab using the genome sequence of an Indian Nipah isolate, it would be easily accessible for commercial purposes in India. In this proposed work, we plan to study host-virus interactions essential for Nipah virus transcription and replication using the minigenome system developed in this study, employing two different approaches. In the first approach, I will identify the RNA-binding proteins that bind to Nipah virus minigenome RNA by performing UV-crosslinking of the virus minigenome RNA and host proteins in live cells, followed by pulling down the viral genomic RNA cross-linked to host proteins using antisense probes. Mass spectrometry will be used to identify the viral genomic RNA-interacting proteins. In the second approach, I plan to perform genome-wide screens with a CRISPR-Cas9 pooled library using the Nipah virus minigenome expressing eGFP to identify host factors involved in viral transcription and replication. Top hits from above proposed studies will be further validated and studied to understand their roles in Nipah virus transcription and replication. In summary, this study would establish a Nipah virus minigenome suitable for BSL-2 facilities using the genome sequence of a Nipah virus isolated in India. Additionally, it would reveal novel host-virus interactions mediating Nipah virus transcription and replication.