LMBD1蛋白質參與非hNTCP依賴性之B型肝炎病毒感染路徑

Abstract

B型肝炎病毒 (hepatitis B virus, HBV)，屬於嗜肝病毒科 (hepadnaviridae)，為具有套模及部分雙股DNA之病毒，其主要為造成人類肝臟疾病之主要原因。目前研究指出人類的鈉依賴性牛磺膽酸鹽共同運輸蛋白質(human Na+-dependent taurocholate co transporting polypeptide；hNTCP)為HBV主要感染之功能性受體。最近的研究中指出HBV大型表面抗原可以被內吞至缺乏表現hNTCP之HepG2細胞中，因此HBV早期感染之機制尚待進一步研究。本實驗室過去的研究指出由LMBRD1基因所編碼的LMBD1蛋白質會與hNTCP有交互作用。此外，在Hus-E/2細胞中當把LMBRD1基因表現減弱(knockdown)，HBV感染Hus-E/2細胞的數量會下降，但當表現過量的LMBD1蛋白質則可以回復HBV感染的現象。因此推測LMBD1會調節HBV的感染。本研究是探討hNTCP與LMBD1蛋白質參與HBV感染過程之角色。qPCR結果顯示在感染HBV 20小時後，缺乏表現hNTCP之HepG2細胞中仍能偵測到HBV基因體DNA。共軛焦顯微鏡分析結果顯示在HepG2細胞感染HBV 20小時後EGFR會與HBV有共位現象。上述結果推測在無hNCTP的情況下HBV仍然能夠被內吞至宿主細胞中。然而在HepG2細胞中感染HBV 72小時後則無法偵測到HBV基因體DNA，只有在添加溶酶體酵素抑制劑chloroquine後才能偵測到HBV基因體DNA。此外，在過量表現hNTCP的HepG2細胞中可以使HBV貼附於細胞上的數量上升了三倍，並且在感染HBV 72小時後仍可偵測到HBV的複製。上述結果猜測hNTCP不只參與在病毒貼附於細胞表面的過程，其可能也參與在HBV從晚期胞內體或溶酶體中釋放到細胞質的脫殼過程。最近的研究指出EGF受體會當作HBV的輔助因子幫助HBV藉由NCTP而被一起內吞至宿主細胞當中。本研究顯示在無hNTCP的情況下，經EGF的誘導HBV就可以被內吞至無血清培養基之HepG2細胞中。此外，共軛焦顯微鏡分析結果也顯示在EGF的誘導下HBV會與EGFR一起內吞至HepG2細胞中。另外利用GST pulldown與免疫沉澱法證實EGFR會與LMBD1及大型表面抗原之pre-S1區域皆具有交互作用。這些結果顯示EGFR可能會參與在非依賴hNTCP介導之內吞作用。為了探討LMBD1蛋白質是否參與在HBV早期感染的過程中，本研究利用帶有LMBRD1 shRNA之慢病毒去減弱HepG2與會表現hNTCP之Hus-E/2細胞中的LMBRD1基因表現。當Hus-E/2細胞中LMBRD1被減弱後，hNTCP表現與HBV貼附於細胞表面數量有顯著性下降。然而在HepG2細胞中，當LMBRD1被減弱後，HBV貼附於細胞表面數量並不會受影響，但是在EGF誘導後HBV內吞至細胞中的數量有顯著性減少。上述結果說明LMBD1蛋白質可能參與在EGF誘導之內吞作用及影響hNTCP的表現量。綜合以上結果，HBV可以透過非依賴性hNTCP及EGFR所介導之內吞作用進入宿主細胞中。而hNTCP會幫助HBV貼附於細胞表面以及可能參與在HBV從溶酶體中釋放到細胞質的脫殼過程。此外，LMBD1可能參與在EGFR介導之內吞作用進而促進HBV的感染。

Hepatitis B virus (HBV) is an enveloped, partially double-strand DNA virus that belongs to the Hepadnaviridae family and is a major cause of human liver diseases. Human hepatic Na+-dependent taurocholate co transporting polypeptide (hNTCP) functions as a receptor for HBV infection. Recently, the large form HBsAg (LHBsAg) was proposed to be involved in the infection of HepG2 cells that lack hNTCP, but the exact mechanism of HBV infection needs to be further studied. Our laboratory has previously demonstrated that LMBD1, encoded by the limb region 1 (LMBR1) domain containing 1 gene (LMBRD1), interacted with hNTCP. In addition, the HBV replication was decreased when LMBRD1 gene was knocked down, and this phenotype could be rescued by overexpression of LMBD1. These data suggest a role of LMBD1 in regulating HBV infection. In this study, the functions of hNTCP and LMBD1 proteins in HBV infection were further investigated. qPCR analysis following HBV infection demonstrated that, without hNTCP expression, HBV genomic DNA could be detected in HepG2 cells at 20-hour postinfection. The result of confocal microscopy further showed that HBV colocalized with LAMP1 in HepG2 cells at 20-hour postinfection. These data indicate that HBV could be internalized into host cells through hNTCP-independent endocytosis. However, there was no HBV genomic DNA was detected in HepG2 cells without hNTCP expression at 72-hour postinfection unless the HepG2 cells were treated with lysosomal enzyme inhibitor chloroquine. In addition, ectopic overexpression of hNTCP increased the attachment of HBV to HepG2 cells by 3-fold and HBV replication was detected at 72-hour postinfection. These data suggest that hNTCP not only facilitates HBV attachment but might also participates in HBV uncoating from the late endosome or lysosome after internalization. Recently, EGFR was demonstrated to be an entry cofactor that mediates HBV-hNTCP internalization into susceptible cells. In this study, HBV internalization into hNTCP-negative HepG2 cells cultured in serum-free medium was detected upon EGF induction. In addition, the result of confocal microscopy further showed that HBV colocalized with EGFR upon EGF induction. GST pulldown and immunoprecipitation assay further showed the interactions of EGFR with LMBD1 and with the pre-S1 domain of LHBsAg. The results indicate that EGFR may involve in hNTCP-independent HBV internalization. To investigate whether LMBD1 participates in HBV infection at early phase, studies were further performed in LMBRD1-knockdown HepG2 cells and the immortalized primary human hepatocytes, Hus-E/2 cells that express hNTCP. The results showed that both the levels of hNTCP expression and HBV attached to the cell surface were decreased in LMBRD1-knockdown Hus-E/2 cells. However, no effect on the attachment of HBV was observed in LMBRD1-knockdown HepG2 cells. Nevertheless, LMBRD1-knockdown reduced the level of EGF-induced HBV internalization into HepG2 cells. These data suggest that LMBD1 participates in the regulation of EGFR-mediated endocytosis and hNTCP expression. In conclusion, HBV could be internalized into host cells through hNTCP-independent EGFR-mediated endocytosis. hNTCP can help HBV to attach on host cell surface and may facilitate HBV uncoating from the lysosome. In addition, LMBD1 might participate in EGFR-mediated endocytosis and thus promotes HBV infection.