Charakterisierung der Interaktion zwischen HBV/HDV und dem spezifischen Rezeptor NTCP durch die Identifizierung von Protein-Protein Interaktionen und Interaktionsdomänen

Beschreibung

Na+/Taurocholate Cotransporting Polypeptide (NTCP, gene symbol SLC10A1) represents the first and best characterized member of the SLC10 carrier family, which currently consists of seven transmembrane proteins. In addition to its physiological function as a hepatic bile acid transporter NTCP was identified as the high affinity entry receptor of the hepatitis B (HBV) and hepatitis D (HDV) viruses. It was shown that homodimerization and heterodimerization within members of the SLC10 carrier family is a typical feature of this protein family. This was experimentally analyzed and confirmed by Membrane Yeast-Two Hybrid (MYTH) experiments in yeast cells and was partially confirmed by co-immunoprecipitation (co-IP) in human HEK293 cells. Protein sequence analysis of the SLC10 proteins revealed both a conserved GlycineXXXGlycine (GXXXG) motif in the seventh, and a partially conserved GXXXG/Alanine (GXXXA) motif in the second transmembrane domain (TMD2/7). Such motifs have previously been analyzed and identified in several other dimerizing proteins. Mutation of these motifs in NTCP resulted in a reduction of dimerization observed in the MYTH-system, which could however not be confirmed by co-IP. Co-localization experiments with wild-type NTCP, as well as transport experiments with taurocholate, confirmed a loss-of-function mutation in the case of the TMD7 mutant. In contrast, the TMD2 mutant behaved quite similar to the wild-type NTCP and showed plasma membrane expression and only slightly decreased transport activity. In addition, a significantly lower HBV-infection rate was shown in cells transfected with the TMD2 NTCP mutant compared with wild-type NTCP. Because dimerization of NTCP is considered an essential factor for successful endocytosis of HBV, it is possible that the TMD2 mutant inhibits HBV-infection due to a lower NTCP dimerization ability. However, the data of the present study suggest that the GXXXG/A motifs of NTCP are more important for correct folding of NTCP and its sorting to the plasma membrane. During MYTH-screening of a kidney cDNA library with the aim of identifying novel protein interaction partners of NTCP, IFITM3 was found as the most dominant hit. IFITM3 plays a role in the innate immunity and is known as a viral restriction factor. In the two human liver cell lines HepG2 and HuH7, the interaction between IFITM3 and NTCP was confirmed by means of co-IP. SiRNA-mediated knockdown experiments confirmed a successful IFITM3-knockdown with high efficiency in hepatoma cells and primary human hepatocytes (PHH). It was found that in the HepG2-cells, decreased IFITM3-expression simultaneously resulted in decreased NTCP-expression at the gene and protein level, as well as a reduced transport rate of taurocholate. Based on this, IFITM3 could act as an NTCP-stabilizing factor. In HuH7-cells and PHH, IFITM3 has been shown to support infection with HBV/HDV. Infection with both viruses was significantly inhibited after knockdown of IFITM3. Due to the fact that the binding of the myr-preS1-peptide under knockdown of IFITM3 did not differ quantitatively from the control, the interaction between IFITM3 and HBV/HDV appears to take place only after binding of HBV/HDV to NTCP. Due to the physiological localization of IFITM3 in the membrane of endolysosomes, it is conceivable that HBV/HDV and IFITM3 interact within the endosomes after endocytosis in such a way that a stronger infection occurs. However, further investigations are needed to uncover the exact mechanism underlying this observation. Based on the data obtained, it seems possible to limit HBV-/ HDV-infection of liver cells by blocking IFITM3, e.g. by rapamycin or amphotericin B. However, blocking IFITM3 would lead to a higher susceptibility of other viral infections which would physiologically be restricted by IFITM3.

Autor*in

Massimo Palatini

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Gallensäure HBV Infektion hepatitis

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