Genetic interrogation of host factors essential to rotavirus infection
BACKGROUND: Rotaviruses (RVs) are the most important etiological cause of severe gastroenteritis and diarrhea among infants and young children on a global scale. RV infection is responsible for at least 215,000 deaths among young children annually, 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. Despite the recent advances in understanding the interaction between RV outer capsid protein VP4 and the host sialic acids (Sia) on cellular surfaces, the molecular details of virus entry into target enterocytes remain unclear.
HYPOTHESIS: Our central hypothesis is that, in contrast to the prevalent notion in the field, human RV infection of enterocytes are dependent on VP4 interaction with specific subtypes of Sia, synthesized and conjugated onto glycoproteins via a Golgi-glycotransferase mediated mechanism.
METHODS: To test our hypothesis, essential genes in Sia synthetic pathways including CMP-sialic acid transporter will be genetically depleted via CRISPR/Cas9 technology in HT-29 cells, a human IEC cell line. RV entry, intracellular replication and production of progeny will be assessed in wild-type and knockout HT-29 cells.
RESULTS: Consistent with our hypothesis, Sia contributed by SLC35A1 signaling mediates human RV infection of HT-29 cells. In addition, we also found that at least another molecule serves as an alternative/redundant human RV receptor in primary human intestinal organoids.
IMPACT: The information from the current study will be invaluable in future screens for small molecule inhibitors or rational design of soluble Sia mimetics that target this pathway to inhibit rotavirus for therapeutic purposes. Our long-term goal is to advance the general knowledge of enteric virus induced diarrhea and help improve global children health.
Website Link: https://med.stanford.edu/profiles/siyuan-ding