Structural Basis for Human Norovirus Capsid Binding to Bile Acids

Abstract

A recently developed human norovirus cell culture system revealed that the presence of bile enhanced or was an essential requirement for the growth of certain genotypes. Before this discovery, histo-blood group antigens (HBGAs) were the only well-studied cofactor known for human noroviruses, and there was evidence that several genotypes poorly bound HBGAs. Therefore, the purpose of this study was to investigate how human norovirus capsids interact with bile acids. We found that bile acids had low-micromolar affinities for GII.1, GII.10, and GII.19 capsids but did not bind GI.1, GII.3, GII.4, or GII.17. We showed that bile acid bound at a partially conserved pocket on the norovirus capsid-protruding (P) domain using X-ray crystallography. Amino acid sequence alignment and structural analysis delivered an explanation of selective bile acid binding. Intriguingly, we discovered that binding of the bile acid was the critical step to stabilize several P domain loops that optimally placed an essential amino acid side chain (Asp375) to bind HBGAs in an otherwise HBGA nonbinder (GII.1). Furthermore, bile acid enhanced HBGA binding for a known HBGA binder (GII.10). Altogether, these new data suggest that bile acid functions as a loop-stabilizing regulator and enhancer of HBGA binding for certain norovirus genotypes. IMPORTANCE Given that human norovirus virions likely interact with bile acid during a natural infection, our evidence that an HBGA nonbinder (GII.1) can be converted to an HBGA binder after bile acid binding is of major significance. Our data provide direct evidence that, like HBGAs, bile acid interaction on the capsid is an important cofactor for certain genotypes. However, more unanswered questions seem to arise from these new discoveries. For example, is there an association between the bile acid requirement and the prevalence of certain genotypes? That is, the GII.1 and GII.10 (bile acid binders) genotypes rarely caused outbreaks, whereas the GII.4 and GII.17 genotypes (bile acid nonbinders) were responsible for large epidemics. Therefore, it seems plausible that certain genotypes require bile acids, whereas others have modified their bile acid requirements on the capsid.

Chemical‐Shift Perturbations Reflect Bile Acid Binding to Norovirus Coat Protein: Recognition Comes in Different Flavors

“…Importantly, for GII.4 Saga P dimers no CSPs have been observed at sites corresponding to two other bile acid binding sites described before . As replication of GII.4 NoVs in human intestinal enteroids has been reported to be significantly enhanced by the presence of bile acids, and because we can exclude binding to one of the putative high‐affinity sites, we suggest that the low‐affinity interaction with viral capsids provides the molecular basis for understanding the role of bile acids in promoting infection.…”

“…Figure S1). NMR signals of amino acids in the HBGA binding pocket or at the sites matching the high‐affinity bile acid binding pockets reported for rare genotypes of human NoV or murine NoV remain unaffected.…”

“…For GII.10 Vietnam026 we expected affinities for bile acids in the micromolar range due to binding to a high‐affinity bile acid binding site adjacent to the HBGA site as described recently . In this case we have no isotope‐labeled P dimers available yet, but we have performed STD NMR experiments with GII.10 Vietnam026 VLPs.…”

“…CA was chosen, as its solubility in water is higher than other bile acids. GCDCA is less water soluble, but was included because GCDCA has been the focus of previous investigations …”

“…Two crystallographic studies addressed the binding of bile acids to human and to murine norovirus capsid protein . For particular strains of human NoVs two symmetrical bile acid binding pockets with affinities in the low‐micromolar range have been identified adjacent to the HBGA binding sites . In the case of a rare GII.1 strain, binding of bile acids promoted attachment to HBGAs.…”