Human hepatitis B virus (HBV) infection and HBV-related diseases remain a major public health problem. Individuals coinfected with its satellite hepatitis D virus (HDV) have more severe disease. Cellular entry of both viruses is mediated by HBV envelope proteins. The pre-S1 domain of the large envelope protein is a key determinant for receptor(s) binding. However, the identity of the receptor(s) is unknown. Here, by using near zero distance photo-cross-linking and tandem affinity purification, we revealed that the receptor-binding region of pre-S1 specifically interacts with sodium taurocholate cotransporting polypeptide (NTCP), a multiple transmembrane transporter predominantly expressed in the liver. Silencing NTCP inhibited HBV and HDV infection, while exogenous NTCP expression rendered nonsusceptible hepatocarcinoma cells susceptible to these viral infections. Moreover, replacing amino acids 157–165 of nonfunctional monkey NTCP with the human counterpart conferred its ability in supporting both viral infections. Our results demonstrate that NTCP is a functional receptor for HBV and HDV.DOI: http://dx.doi.org/10.7554/eLife.00049.001
We also found that gene expression patterns can be used to predict the aggressiveness of prostate cancer using a novel model.
The liver bile acids transporter sodium taurocholate cotransporting polypeptide (NTCP) is responsible for the majority of sodium-dependent bile salts uptake by hepatocytes. NTCP also functions as a cellular receptor for viral entry of hepatitis B virus (HBV) and hepatitis D virus (HDV) through a specific interaction between NTCP and the pre-S1 domain of HBV large envelope protein. However, it remains unknown if these two functions of NTCP are independent or if they interfere with each other. Here we show that binding of the pre-S1 domain to human NTCP blocks taurocholate uptake by the receptor; conversely, some bile acid substrates of NTCP inhibit HBV and HDV entry. Mutations of NTCP residues critical for bile salts binding severely impair viral infection by HDV and HBV; to a lesser extent, the residues important for sodium binding also inhibit viral infection. The mutation S267F, corresponding to a single nucleotide polymorphism (SNP) found in about 9% of the East Asian population, renders NTCP without either taurocholate transporting activity or the ability to support HBV or HDV infection in cell culture. These results demonstrate that molecular determinants critical for HBV and HDV entry overlap with that for bile salts uptake by NTCP, indicating that viral infection may interfere with the normal function of NTCP, and bile acids and their derivatives hold the potential for further development into antiviral drugs. IMPORTANCE Human hepatitis B virus (HBV) and its satellite virus, hepatitis D virus (HDV), are important human pathogens. Available therapeutics against HBV are limited, and there is no drug that is clinically available for HDV infection. A liver bile acids transporter (sodium taurocholate cotransporting polypeptide [NTCP]) critical for maintaining homeostasis of bile acids serves as a functional receptor for HBV and HDV. We report here that the NTCP-binding lipopeptide that originates from the first 47 amino acids of the pre-S1 domain of the HBV L protein blocks taurocholate transport. Some bile salts dose dependently inhibit HBV and HDV infection mediated by NTCP; molecular determinants of NTCP critical for HBV and HDV entry overlap with that for bile acids transport. This work advances our understanding of NTCP-mediated HBV and HDV infection in relation to NTCP's physiological function. Our results also suggest that bile acids or their derivatives hold potential for development into novel drugs against HBV and HDV infection.
H epatocellular carcinomas (HCC) and hepatoblastomas of childhood (HPBL) are two types of liver cancer with high mortality and morbidity and international prevalence. There have been several recent studies of patterns of gene expression and molecular classification of HCC. [1][2][3][4] The studies demonstrated that HCC can be clustered in subgroups of gene expression patterns that have different prognostic and clinical behavior. Other recent studies also examined similarities between HCC precursor lesions (low and high grade liver nodules) and demonstrated significant similarities but also differences between HCC and precursor lesions. 5 In this study, we also focused on gene expression of HCC and HPBL, but from a different perspective than previous studies. We utilized a set of tissues from normal liver (NL), HCC, HPBL and tumor adjacent (AT) tissues and determined gene expression patterns not as a ratio of tumor vs. normal, but rather as absolute separate values for each unique tissue. This allowed standard but stringent statistical analysis not feasible when gene expression is only viewed as a fold change over normal tissues. Identification of gene expression patterns of liver tumors from this perspective allows identification of the main differences between the tumor subtypes and the adjacent nontumor (but often cirrhotic) liver; it also offers the potential of defining new therapeutic and diagnostic modalities. Our findings include some genes already shown to increase in HCC, thus validating our overall approach. Our results also revealed many other genes, not so far involved with biology of liver tumors. In addition, we carried a whole genome analysis of 27 HCC and determined chromosomal loci with genetic abnormalities common to most of the HCC. Materials and MethodsSee Supplemental information at the HEPATOLOGY
Human hepatitis B virus (HBV) and its satellite virus, hepatitis D virus (HDV), primarily infect humans, chimpanzees, or tree shrews (Tupaia belangeri). Viral infections in other species are known to be mainly restricted at the entry level since viral replication can be achieved in the cells by transfection of the viral genome. Sodium taurocholate cotransporting polypeptide (NTCP) is a functional receptor for HBV and HDV, and amino acids 157 to 165 of NTCP are critical for viral entry and likely limit viral infection of macaques. However, the molecular determinants for viral entry restriction in mouse NTCP (mNTCP) remain unclear. In this study, mNTCP was found to be unable to support either HBV or HDV infection, although it can bind to pre-S1 of HBV L protein and is functional in transporting substrate taurocholate; comprehensive swapping and point mutations of human NTCP (hNTCP) and mNTCP revealed molecular determinants restricting mNTCP for viral entry of HBV and HDV. Remarkably, when mNTCP residues 84 to 87 were substituted by human counterparts, mNTCP can effectively support viral infections. In addition, a number of cell lines, regardless of their species or tissue origin, supported HDV infection when transfected with hNTCP or mNTCP with residues 84 to 87 replaced by human counterparts, highlighting the central role of NTCP for viral infections mediated by HBV envelope proteins. These studies advance our understanding of NTCP-mediated viral entry of HBV and HDV and have important implications for developing the mouse model for their infections. Hepatitis B virus (HBV) is the prototype of the Hepadnaviridae (hepatotropic DNA viruses) family (1). Human HBV has infected 2 billion people worldwide, and 350 million of them are chronically infected (2). About two-thirds of hepatocellular carcinoma (HCC) is due to chronic HBV infection (3). Hepatitis D virus (HDV) is a satellite virus of HBV, 15 million people are infected by HDV, and no specific anti-HDV drug is clinically available at present. Chronic HBV patients coinfected with HDV are at high risk for more severe symptoms and more rapid progression (4).HBV is a small enveloped virus with a relaxed circular partially double-stranded DNA genome of ϳ3.2 kb encoding four overlapped open reading frames. HBV large (L), middle (M), and small (S) envelope proteins are encoded by a single open reading frame (5). They are translated from different initial codons but share an end. HDV contains a single-stranded, circular RNA genome of ϳ1,700 nucleotides, with one coding region for small and large form of delta antigens. It replicates in the nucleus and accumulates a large number of viral RNAs and delta antigen (6). Since HDV has to employ HBV envelope proteins for the infection of hepatocytes (7), the entry of HDV is believed to be similar to that of HBV and has been used as a surrogate to study the early entry process (4,8,9).The lack of a convenient in vitro viral infection system has been a long-standing hurdle for studying viral entry of HBV and HDV (10). Recently, ...
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