A Farewell to Arms: Breaking the Species Barrier of Hepatitis B Virus in Small Non-human Primates

Abstract

Hepatitis B virus (HBV) remains a major cause of liver disease in humans, affecting approximately 257 million people worldwide. Despite the availability of treatments for chronic HBV, a cure remains elusive. As novel therapeutics to combat HBV persistence are being developed, animal models are urgently needed to evaluate their efficacy and safety. The narrow host tropism of HBV remains a challenge in establishing a long-lasting HBV infection model in vivo, but recent studies have provided optimism toward a small nonhuman primate (NHP) model. Previous research has shown that HBV restriction in NHP species is primarily attributed to incompatibilities between the preS1 region of the viral envelope protein and the cellular functional receptor – sodium taurocholate co-transporting peptide (NTCP) – in non-permissive primates. Particularly, amino acids 157-165 in Old World monkey (OWM) NTCPs appear to be incompatible with the viral envelope. NTCP from the cynomolgus macaque (CynoNTCP), an OWM, was manipulated in this study to investigate the mechanism of HBV restriction in OWM at the entry level. Since the humanizing substitution R158G on CynoNTCP has been found to be necessary for HBV binding and uptake when expressed on HepG2 cells, CynoNTCP variants with humanized mutations in combination with the R158 mutation were constructed as follows: G157K, I160V, L161I, and P165L. To investigate the role of these residues in virion uptake, HBV preS1 peptides conjugated with FITC fluorophore were used for imaging, and flow cytometry to determine the binding ability of CynoNTCP mutants. Additionally, an HBV infection assay was conducted on HepG2 cell lines expressing the above CynoNTCP variants. The results revealed that the G157K & R158G variant reduced the ability of CynoNTCP to mediate binding and uptake of HBV virions, whereas the R158G & P165L variant enhanced infection levels significantly. This suggests that 157K may play negative role in HBV infection, while 165L is another important residue in NTCP that can confer or enhance HBV susceptibility. This novel information sheds insight into the evolutionary strategies of HBV resistance that were established during the emergence of OWM, and it may aid future studies that aim to expand HBV tropism and build a small NHP model to bolster tools for studying HBV in vivo.