RT Journal Article SR Electronic(1) A1 Goddard, Chun A1 Irving, William A1 Tarr, AlexanderYR 2019 T1 Optimisation of an in vitro cell system using pseudoviruses to investigate HBV entry mechanisms JF Access Microbiology, VO 1 IS 1A OP SP 204 DO https://doi.org/10.1099/acmi.ac2019.po0076 PB Microbiology Society, SN 2516-8290, AB Aim To explore the roles of large (L), middle (M) and small (S) surface antigens in an in vitro model of hepatitis B virus (HBV) entry, and thereby to achieve an optimal in vitro cell system using pseudoviruses to investigate HBV entry mechanisms. Methods RNA encoding sodium taurocholate co-transporting polypeptide (NTCP) was extracted from human hepatocytes and cloned into the pHIV-EGFP expression vector. The resulting pHIV-NTCP-EGFP construct was delivered into Huh7 hepatoma cells with the aid of pCMVR87.4 (packaging vector) and pCMV.VSV.G (glycoprotein), subsequently, a cell line over-expressing NTCP was generated. Meanwhile, by silencing start codons at L, M or S, seven constructs were obtained, i.e. L + M -S-, L-M + S -, l -M-S+, L + M + S-, L-M + S +, L + M -S+, l -M-S-. A matrix with various amounts of the seven constructs was used to generate HBV pseudoparticles (HBVpp) using a Luciferase-based HIV (pNL4.3.luc.R-E-) pseudotype entry model system. The infectivity of the HBVpp was tested in NTCP naïve and NTCP over-expressing Huh7 hepatoma cells. Results The relative amounts of L, M and S were critical in determining the efficiency of entry of HBVpp into NTCP + ve Huh7 cells. Conclusions Creation of NTCP-over expressing cells together with optimisation of conditions to maximise HBVpp entry provides an important tool to investigate the entry step in the HBV life cycle, and may allow identification of non-NTCP-dependent viral entry pathways. , UL https://www.microbiologyresearch.org/content/journal/acmi/10.1099/acmi.ac2019.po0076