Concentration of Na+-taurocholate-cotransporting polypeptide expressed after in vitro-transcribed mRNA transfection determines susceptibility of hepatoma cells for hepatitis B virus

Abstract

Infection of hepatocytes by hepatitis B virus (HBV) depends on surface expression of its receptor Na+-taurocholate-cotransporting polypeptide (NTCP), but sufficient NTCP expression is lacking in most cell lines. NTCP can be introduced by plasmid transfection or transduction by viral vectors to render cells permissive for HBV. However, transient transfection of hepatocyte-derived cell lines is inefficient, resulting in inhomogeneous protein expression and does not allow to adapt the level of NTCP expression. We therefore utilized in vitro transcribed mRNA to introduce NTCP into cells. Optimization using alternative cap structures and nucleotide modifications rendered mRNA transfection into different non-hepatic and hepatic cell lines very efficient. After transfection of mRNA, surface expression and functionality of NTCP was demonstrated by staining with an N-terminal HBV-preS peptide and bile acid uptake. Introduction of NTCP by mRNA transfection increased susceptibility of hepatoma cells to HBV in a dose-dependent manner. Transfection of NTCP mRNA into non-liver cells, in contrast, supported bile acid uptake but did still not render the cells permissive for HBV, demonstrating the requirement for additional host factors. Introduction of candidate host factors by mRNA transfection will allow for fast and convenient analysis of the viral life cycle using a transient, but reliable expression system.

Host cell-dependent late entry step as determinant of hepatitis B virus infection

“…We previously reported that the human embryonic kidney cell line HEK293 supports HBV and DHBV cccDNA formation via intracellular amplification [ 8 , 42 ]. However, others have reported that HEK293 cells failed to support productive HBV infection, as determined by the lack of detectable HBV antigen expression [ 23 ]. To test whether HEK293 cells indeed behave similar to WC3 cells, i.e., supporting cccDNA formation via intracellular amplification but not infection after huNTCP expression, we established huNTCP-expressing HEK293 cells (HEK293-huNTCP) by lentiviral transduction.…”

“…Importantly, in this study, we detected HBV cccDNA in the huNTCP-expressing human non-hepatic HEK293 and woodchuck WCH-17 cells during infection, suggesting that neither human- nor hepatocyte-specific factors are required for HBV cccDNA formation via de novo infection. Of course, HEK293 cells remain unable to support productive HBV replication after cccDNA formation due to the lack of liver transcription factors that are essential for HBV cccDNA transcription [ 24 , 45 ], which explains the inability of HEK293 cells to allow productive HBV replication after huNTCP expression in a recent study [ 23 ]. On the other hand, our results indicated that human-specific hepatocyte transcription factors are not essential determinants for HBV species tropism as all HBV promoters did show activity at varying degrees in the woodchuck hepatic cells.…”

“…On the other hand, previous studies, which used HBV antigen expression and/or secretion as readouts for successful HBV infection, indicated that non-hepatic cells (e.g., HeLa, HEK293, A549, U2OS, CHO, and Vero cells) expressing huNTCP cannot support HBV infection [ 15 , 16 , 23 ], although they can support HDV infection [ 15 , 16 ]. It is well-known that liver specific or enriched transcription factors, which are required for HBV gene transcription, are critical determinants of HBV tissue tropism [ 24 ].…”

Presence of entry receptors and viral markers suggest a low level of placental replication of hepatitis B virus in a proportion of pregnant women infected with chronic hepatitis B

“…For this to happen, placenta must express entry receptor(s) for HBV. NTCP is the primary receptor for virus on hepatocytes and is thought to be exclusive to these cells 12 . We looked for the expression data of NTCP in different tissues/cells available in "The Human Protein Atlas" ( http://www.proteinatlas.org ).…”

Hepatitis B Virus Capsid: The Core in Productive Entry and Covalently Closed Circular DNA Formation

“…This result, together with the fact that HEK293 cells can also support cccDNA formation via intracellular amplification [ 11 , 81 ], indicates that hepatocyte-specific factors are also not essential for nucleocapsid uncoating. Of course, non-hepatic cells, even after huNTCP expression, will remain unable to support the full HBV lifecycle as HBV gene expression cannot take place due to the lack of hepatocyte-specific transcription factors, which are necessary for cccDNA transcription and serve as additional host determinants of hepatocyte tropism of HBV [ 71 , 82 , 83 ]. For example, the NIH 3T3 fibroblasts were unable to support viral replication (pgRNA synthesis or rcDNA production) unless the hepatocyte-specific (enriched) transcription factors HNF4, RXRα, and PPARα were exogenously supplied, demonstrating that HBV replication is dependent on liver-specific transcription factors [ 83 ].…”