Mechanistic Investigation of a Rotavirus Auxiliary Protein NSP6 Countering Host Defense

Rotaviruses (RVs) are the leading infectious agent of severe gastroenteritis in infants and young children. RVs encode a small non-structural protein 6 (NSP6) with no known function. Studying NSP6 function is difficult as it is expressed as a frameshifted product from a double-stranded RNA gene segment that also encodes another RV protein NSP5 and the two open reading frames completely overlap. To overcome these hurdles, we developed a robust RV reverse genetics system and generated a recombinant NSP6-deletion RV with all possible NSP6 start codons removed without affecting NSP5 expression. For the first time, we have the opportunity to examine the physiological role of NSP6 in RV replication, pathogenesis, and immunity in organoid cultures and in vivo. In preliminary studies, we found that in neonatal mice, oral inoculation of an NSP6-deletion RV resulted in significantly lower intestinal replication and fewer diarrhea incidences than the parental virus starting at 6 days post infection. Single-cell RNA-sequencing, further validated by multi-color flow cytometry, revealed that NSP6 expression correlated with reduced T cell numbers at the site of RV infection. NSP6 was secreted from RV- infected intestinal epithelial cells and blocked chemotactic migration of T cells in a transwell assay. Our long- term goal is to delineate a novel immune evasion role of RV NSP6 and use the knowledge towards preventing and treating RV infection. In aim 1, we hypothesize that a predicted four-α-helix structure of NSP6 is critical for its immunoinhibitory activities. We aim to: (1) define the key amino acids within NSP6 responsible for secretion and chemoattractant binding; and (2) identify how NSP6 sequence variation between human and animal RV strains regulates virulence and zoonotic infection. We expect that critical mutations in α-helices will disrupt secretion and that truncated NSP6 proteins from porcine and attenuated human RV strains cannot functionally replace the full- length murine RV NSP6 protein in vivo. In aim 2, we hypothesize that RV NSP6 is a novel virulence factor that acts as a molecular mimicry of chemokine receptors and inhibits T cell recruitment. We aim to: (1) characterize the effect of NSP6 on systemic and local intestinal CD4+ and CD8+ T cell responses; (2) identify the migratory patterns of T cells towards RV-infected epithelial cells by two-photon microscopy; and (3) determine the immunogenicity and protective efficacy of an NSP6-deletion RV as a vaccine candidate. We expect that NSP6 is secreted basolaterally from infected intestinal epithelial cells and suppresses T cell chemotaxis. We also expect that immunization with an NSP6-deficient RV will induce cross-reactive T cell responses and confer heterotypic immunity against subsequent challenges. By interrogating RV NSP6-T cell interactions, we will gain new information that aids in development of improved live-attenuated RV vaccines and design of novel therapeutics that dampen undesired T cell-mediated inflammation in the intestine. Project Number: 1R01AI189930-01 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: Siyuan Ding | Institution: WASHINGTON UNIVERSITY, SAINT LOUIS, MO | Award Amount: $593,021 | Activity Code: R01 | Study Section: Viral Pathogenesis and Immunity Study Section [VPI] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R01AI18993001