The SRL peptide of rhesus rotavirus VP4 protein governs cholangiocyte infection and the murine model of biliary atresia

Abstract

Biliary atresia (BA) is a neonatal obstructive cholangiopathy which progresses to end stage liver disease, often requiring transplantation. The murine model of BA, employing rhesus rotavirus (RRV), parallels human disease and has been used to elucidate mechanistic aspects of a virus induced biliary cholangiopathy. We previously reported that RRV VP4 gene plays an integral role in activating the immune system and induction of BA. Utilizing rotavirus binding and blocking assays, this study elucidated how RRV VP4 protein governs cholangiocyte susceptibility to infection both in vitro and in vivo in the murine model of BA. We identified the amino acid sequence on VP4 and its cholangiocyte binding protein, finding that the sequence is specific to those rotavirus strains which cause an obstructive cholangiopathy. Pretreatment of murine and human cholangiocytes with this VP4 derived peptide (TRTRVSRLY), significantly reduced RRV’s ability to bind and infect the cells. However, the peptide did not block cholangiocyte binding of TUCH and Ro1845, strains which do not induce murine BA. The SRL sequence within TRTRVSRLY is required for cholangiocyte binding and viral replication. The cholangiocyte membrane protein bound by SRL was found to be Hsc70. Inhibition of Hsc70 by siRNAs reduced RRV’s ability to infect cholangiocytes. This virus-cholangiocyte interaction is also seen in vivo in the murine model of BA, where inoculation of mice with TRTRVSRLY peptide significantly reduced symptoms and mortality in RRV-injected mice. Conclusion The tri-peptide SRL on RRV VP4 binds to the cholangiocyte membrane protein Hsc70 defining a novel binding site governing VP4 attachment. Investigations are underway to determine the cellular response following this interaction to understand how it contributes to the pathogenesis of BA.

Innate Immunity and Pathogenesis of Biliary Atresia

“…Further research led to the identification of a key amino acid sequence "SRL" in VP4, a sequence specific to those rotavirus strains that cause obstructive cholangiopathy (57). This tripeptide "SRL" on RRV VP4 was found to bind specifically to the cholangiocyte membrane protein heat shock cognate 70 (Hsc70), defining a novel binding site governing VP4 attachment (57). To gain insight into the mechanisms involved upon VP4-mediated infection, a reverse genetics system was developed to create a mutant of RRV with a single amino acid change in the VP4 protein and compared to that of wild-type RRV (where the arginine "R" in "SRL" region was replaced with glycine "G") (58).…”

A Point Mutation in the Rhesus Rotavirus VP4 Protein Generated through a Rotavirus Reverse Genetics System Attenuates Biliary Atresia in the Murine Model

“…The basis for the reduction in replication is unclear; it may be a function of binding and entry but also may be a factor of how a virus traffics intracellularly following entry and how this, in turn, triggers the host immune response. Our previous data support this possibility, as we found that binding of arginine (R) to Hsc70 was required (33), which in turn governed translocation of RRV inside the cell. Alternatively, the mutant virus may not infect immune cells (dendritic cells), which also governs how the host immune system responds and has been shown to be a crucial step required for the development of BA (49).…”

“…We recently demonstrated that RRV utilizes the SRL region of its VP4 protein to bind to extracellular Hsc70 (33). Through blocking assays, we demonstrated that the peptide VSRLY is capable of blocking RRV's ability to bind to cholangiocytes.…”

“…Subsequent studies demonstrated that the peptide TRTRVSRLY within the VP4 protein was able to block RRV's ability to bind to and infect cholangiocytes (33), and when this peptide was injected into mice in vivo, it could attenuate the disease phenotype. Furthermore, we determined that the 3-amino-acid sequence SRL within this region, most importantly the amino acid arginine (R), plays a critical role.…”

“…Our previous studies showed that the VP4 gene of RRV is the major determinant in modulating disease pathogenesis (31,32). We recently found that the VP4 gene plays a role in primary binding to cells (cholangiocytes and MA104 cells) prior to entry and that treating cells with the VP4-derived peptide TRTRVSRLY (amino acids 440 to 449 of VP4) can inhibit virus binding and subsequent replication (33). In that study, we identified the specific amino acid sequence SRL within TRTRVSRLY and determined that it binds to Hsc70, driving cell entry.…”