Project Details
Early Career
Status: Funded - Closed
Genetic interrogation of host factors essential to rotavirus infection
Summary
BACKGROUND: Rotaviruses (RVs) are the most important etiological cause of acute gastroenteritis, dehydration, and diarrhea among infants and young children. RV infection is responsible for at least 215,000 deaths annually on a global scale, the majority of them under the age of five and occurring in less developed countries. GAP: RV predominantly infects the absorptive enterocytes in the small intestines of the host species. RV natural infection or vaccination is able to induce human mAbs that efficiently neutralize multiple RV strains. However, the molecular details of virus entry and how that process is blocked by neutralizing antibodies remain unclear. HYPOTHESIS: Our central hypothesis is that, in contrast to the prevalent notion in the field that human RVs bind to HBGA group, human RV infection of enterocytes are dependent on VP4 interaction with a specific yet-to-be-identified receptor, and such interaction is blocked by human mAbs that are directed against VP4 N-terminal region. METHODS: To test our hypothesis, we employed the CRISPR/Cas9 technology to genetically delete essential genes in sialic acid synthetic pathways including CMP-sialic acid transporter in HT-29 cells. RV entry, intracellular replication and production of progeny will be assessed in wild-type and respective knockout HT-29 cells. RESULTS: Human mAbs that we isolated in a prior study efficiently neutralized human RVs in HT-29 cells and primary human intestinal enteroids but not in MA104 cells. We also found that the antibody binding site on RV VP4 is distant from the previous characterized HBGA locus, suggesting that at least another molecule serves as an alternative/redundant human RV receptor in primary human intestinal epithelial cells. IMPACT: The information from the current study will be highly invaluable in designing future clinical studies that survey neutralization activities in human serum that efficiently block human RV infection. Since MA104 cells are the most commonly used cell line to detect anti-RV neutralization activity, our findings suggest that prior vaccine and other studies of human RV neutralization responses may have underestimated the contribution of VP8* antibodies to the overall neutralization titer. Therefore, we propose that human intestinal epithelial cell-based neutralization assays will more truthfully reflect the neutralization titers in human serum. Our long-term goal is to advance the general knowledge of enteric virus induced diarrhea and help improve global children health. Website Link: https://sites.wustl.edu/dinglab/
Publications:
Song Y*, Feng N*, Sanchez-Tacuba L, Yasukawa LL, Ren L, Silverman RH, Ding S#, and Greenberg HB#. Reverse genetics reveals a role of the rotavirus VP3 phosphodiesterase activity in inhibiting RNase L signaling and contributing to intestinal viral replication in vivo. (2019) J Virol. In Press. * co-first authors, # co-corresponding authors