Research Projects

Persistence of RNA viruses in host tissues has come to light with recent studies related to the emergence of new variants of concern of SARS-Cov2. Salivary glands have been discovered to be the niche for viral persistence for SARS-Cov2 and enteric viruses like Rotavirus (RV), and Norovirus from the research of our laboratory and others. Previously it was believed that RNA viruses after completing their life cycles within a host cell, exit by cellular lysis or get neutralized by the immune response of the host. Thus persistence of RNA viruses has remained a virgin field of research. In the current proposal, we attempt to unravel one of the mechanisms that help RNA viruses persistently propagate in salivary gland epithelial cells. We have divided our proposal into 2 specific objectives- Objective 1: Identifying sncRNAs sorted with RV inside microvesicles regulating Interferon- lambda: RVs have been previously reported to transmit inside microvesicles (vesicle size- 200-1000nm). In addition to virions, small non-coding RNAs (sncRNAs) are sorted inside these microvesicles, whose role in viral propagation is still enigmatic. We hypothesize that these sncRNAs may have regulatory roles in the host immune response and other metabolic pathways. IFN-lambda play a major role in interfering with viral invasion and persistence in epithelial cells of mucosal tissues including salivary glands. We plan to isolate RV-containing vesicles from patient samples and cell culture supernatant from salivary gland organoid culture and sequence the contents, to identify the sncRNAs present in them. Thereafter, mapping them to find out their interactions with IFN-lambda. The sncRNAs having a role in inhibiting IFN-lambda response and facilitating viral persistence will be validated in-vitro. Objective 2: Deciphering the sorting mechanism of RV with sncRNA inside microvesicles: Although we know RVs get packaged inside microvesicles, the sorting mechanism that governs the selection of the sncRNAs along with RV virions is not known. Using NSP4 one of the non-structural proteins of RV that plays role in viral assembly in the final stages of RV life cycle, we will try to identify its interacting partners through Mass Spectrometry. The identified proteins will hence map for their role in Endosomal Sorting Complex Required for Transport (ESCRT)-dependent or independent pathway for sorting into microvesicles. With the help of sequential knockdown experiments, the mechanism of sorting will be studied in-vitro. RV infection is one of the dreaded pediatric diseases that still accounts for hospitalization in neonatal intensive care units. Persistence of this virus in salivary glands spills out infectious strains of the virus which results in outbreaks. Identifying the sncRNAs and the mechanism by which they are packaged inside vesicles along with RVs will provide new anti-viral targets that will help in eliminating the virus from the tissue and hence, the persistence.