The C-terminal region of the hepatitis B virus X protein is required for its stimulation of HBV replication in primary mouse hepatocytes

Hepatitis B virus (HBV) infection is an important risk factor for hepatocellular carcinoma (HCC). The hepatitis B virus X protein (HBx), a multifunctional regulatory protein encoded by HBV, is known to be involved in stimulation of viral replication by modulating cell cycle status. HBx is required for maximal virus replication in plasmid-based replication assays in immortalized human liver HepG2 cells and in primary rat hepatocytes. Moreover, the C-terminal region of HBx is important for HBV replication in HepG2 cells. However, in normal hepatocytes, the region of HBx that is responsible for its effect on cell cycle regulation and HBV replication is unclear. We have demonstrated that HBx is similarly required for maximal HBV replication in primary mouse hepatocytes and that the C-terminus of HBx is essential for its ability to stimulate HBV replication by inducing quiescent hepatocytes to exit G0 phase of the cell cycle but stall in G1 phase. Our studies establish that primary mouse hepatocytes support HBx-dependent HBV replication, and provide further evidence for the effect of the C-terminal region of HBx on HBV infection and replication.     

The C-terminal half of the preS1 region is essential for the secretion of human hepatitis B virus large S protein devoid of the N-terminal retention sequence

A large surface protein of human hepatitis B virus was expressed from a mutated S gene in which both the reported retention sequence (aa 2-19) and the C-terminal half of the preS1 region (aa 66-117) were deleted. This retention sequence-free protein was not secreted from the monkey kidney cell line COS-M6 in transient expression assays. When this large S protein was overexpressed in the presence of the major S protein, the secretion of the latter was severely inhibited. Moreover, the presence of S protein in large abundance did not facilitate the secretion of the mutated large S protein at all, indicating that it was subject to more profound retention than the wild-type large S protein. The findings that, like the wild-type large S protein, this altered protein retained some of the properties of its C-terminal S domain, and that the preS1 region was still on its surface suggested that there was no extensive alteration of the polypeptide folding. The interpretation is favored that the C-terminal half of the preS1 region plays a crucial role in the secretion of the large S protein when its N-terminal retention sequence is not present.     

Processing of hepatitis C virus core protein is regulated by its C-terminal sequence

Polyprotein processing of plus-strand RNA viruses is important in the regulation of gene production and replication. The core protein of hepatitis C virus (HCV), constructing the viral particle, is processed from its precursor polyprotein and observed as two forms, p23 and p21. Production of p21 by cleavage at the C-terminus of p23 is considered crucial to viral assembly and replication. In this study, this processing step was compared between clones isolated from two patients with fulminant hepatitis and from five patients with chronic hepatitis by an in vitro translation assay and cell transfection assay. The p21 core protein was predominant from the clone isolated from one of the fulminant hepatitis patient (p21 core protein production was 65.98%), while p23 was abundant with clones from five chronic hepatitis patients (p21 core protein production was 7.11+/-1.62%) and clone from another fulminant hepatitis patient (p21 core protein production was 13.36%). Investigations with chimeric and mutation-introduced constructs revealed that four amino acid residues in the C-terminus of the core region are responsible for this difference. The data suggest that core protein processing is regulated by C-terminus mutations.     

Myristylation of a duck hepatitis B virus envelope protein is essential for infectivity but not for virus assembly

In addition to the major surface (S) protein, the envelope of the duck hepatitis B virus (DHBV) contains a related presurface (preS) protein whose N-terminus bears a covalently attached myristate group. We have explored the functional significance of this modification by examining the replicative potential of a mutant viral genome whose myristylation signal has been inactivated. Following transfection into permissive hepatoma cells, the mutant expresses an unmyristylated preS protein of normal size, immunoreactivity and stability. Cytoplasmic cores containing viral DNA are synthesized, and Dane particles are assembled and exported into the medium. However, the mutant is noninfectious when inoculated into susceptible ducklings. We conclude that myristylation of preS proteins is essential for hepadnaviral infectivity but not for viral assembly; myristylation is most likely required for an early step of the life cycle involving the entry or uncoating of virus particles.     

Mutations of the X region of hepatitis B virus and their clinical implications

Nucleotide (nt) sequences of the X region of more than 130 hepatitis B virus (HBV) Isolates were determined and derived from patients with a variety of clinical features. Correlation of not substitutions with clinicopathological characteristies was attempted. The X region (465nt) Is crucial for the replication and expression of HBV because the X protein trans-activates the HBV genes and this region contains the core promoter, enhancer II, and two direct repeats. There are several mutational hotspots, some of which seem to relate to immunological epitopes of the X protein. Two kinds of mutations which have important clinical significances were found. One is an 8-nt deletion between nt 1770 and 1777, which truncates 20 amino acids from the carboxyl terminus of the X protein. This deletion leads to the suppression of replication and expression of HBV DNA, resulting In immunoserological marker (HBsAg) negativity. This silent HBV infection Is responsible for the majority of non A to non-E hepatitis. The other mutation substituting T for C (nt 1655), T for A (nt 1764) and A for G (nt 1766) seems to relate to fulminant hepatitis. Further sequencing studies and In vitro mutagenesis experiments will clarify the significance of other mutations of the X region.     

GTPase activity is not essential for the interferon-inducible MxA protein to inhibit the replication of hepatitis B virus

Multiple studies have established that GTPase activity is critical for MxA to act against RNA viruses. Recently, it was shown that MxA can also restrict the replication of hepatitis B virus (HBV), a DNA virus, but the requirements for GTPase activity in inhibition of HBV by MxA remain unknown. Here, we report that GTPase-defective mutants (K83A, T103A, and L612K) can downregulate extracellullar HBsAg and HBeAg and reduce the expression of extra- and intracellular HBV DNA in HepG2 cells to levels similar to that achieved by wild-type MxA. Furthermore, TMxA and T103, two nuclear forms of wild-type MxA and a GTPase-defective mutant (T103A) could only slightly decrease the expression of extra- and intracellular HBV DNA in HepG2 cells. In conclusion, GTPase activity is not essential for MxA protein to inhibit HBV replication, and MxA may have only a minimal effect on the replicative cycle of HBV in the nucleus.     

Increased liver pathology in hepatitis C virus transgenic mice expressing the hepatitis B virus X protein

Transgenic mice expressing the full-length HCV coding sequence were crossed with mice that express the HBV X gene-encoded regulatory protein HBx (ATX mice) to test the hypothesis that HBx expression accelerates HCV-induced liver pathogenesis. At 16 months (mo) of age, hepatocellular carcinoma was identified in 21% of HCV/ATX mice, but in none of the single transgenic animals. Analysis of 8-mo animals revealed that, relative to HCV/WT mice, HCV/ATX mice had more severe steatosis, greater liver-to-body weight ratios, and a significant increase in the percentage of hepatocytes staining for proliferating cell nuclear antigen. Furthermore, primary hepatocytes from HCV, ATX, and HCV/ATX transgenic mice were more resistant to fas-mediated apoptosis than hepatocytes from nontransgenic littermates. These results indicate that HBx expression contributes to increased liver pathogenesis in HCV transgenic mice by a mechanism that involves an imbalance in hepatocyte death and regeneration within the context of severe steatosis.     

Characterization and gene cloning of monoclonal antibody specific for the hepatitis B virus X protein

The hepatis B virus X protein (HBx) has been thought to be implicated in the development of hepatocellular carcinoma. Although many functions of HBx have been reported, it is not clear which of HBx functions is important in hepatocellular carcinogenesis. To study HBx function, we produced a monoclonal anti-HBx Ab secreted by hybridoma cell clone H7 and mapped its epitope to a region of HBx between amino acids 29 and 48 by Western blot with truncated forms of HBx and by enzyme-linked immunoadsorbent assay (ELISA) with synthetic HBx peptides. The variable regions of H7 anti-HBx Ab were cloned by polymerase chain reaction using the degenerate-primers and by the 5' rapid amplification-cDNA end method. The sequence analyses revealed that the variable gene segments of the heavy and light chains are the members of mouse heavy chain variable gene 1 family and kappa light chain variable gene 2 family, respectively. In addition, J(H)2 or Jkappa4 gene segment at the end of the heavy-chain or light-chain variable region and DSP2.x gene segment in the CDR 3 of heavy chain were identified.     

An internal domain of the hepatitis B virus X antigen is necessary for transactivating activity

A number of deletion mutants of the hepatitis B viral X antigen had been constructed and assayed for their ability to transactivate heterologous viral regulatory elements. Neither the N-terminal nor the C-terminal amino acid residues were required for transactivating activity. Transactivating activity that resided within amino acid residues 32 to 148 of the X antigen did not show any target DNA sequence or cell line specificity.     

The Vero cell receptor for the hepatitis B virus small S protein is a sialoglycoprotein

The Vero (African green monkey kidney-derived) cell line is capable of binding recombinant hepatitis B surface antigen (rHBsAg) particles containing only the small surface (S) protein of hepatitis B virus (HBV). This binding activity appears to be due to a single major population of receptors (M. E. Peeples et al., Virology 160, 135-142 (1987]. Since infectious HBV particles also contain the small S protein, it is possible that the Vero cell receptor might also function as an HBV receptor. The initial physical characterization of this receptor is reported here. Treatment of Vero cells with each of four proteases reduced their binding activity by 70% or greater, indicating that the receptor is partially protein in nature. Binding activity was also reduced by pretreating cells with neuraminidase or low levels of sodium periodate, indicating that sialic acid also plays a major role in the receptor activity. Consistent with this interpretation, N-acetylneuraminic acid and N-acetylneuraminyl-lactose were able to competitively inhibit rHBsAg particle attachment to Vero cells. The protein nature of the Vero cell receptor was confirmed by the demonstration that chymotrypsin treatment which resulted in 70% loss of binding had little effect on the cell sialic acid content. Therefore, the Vero cell receptor for rHBsAg particles is a sialoglycoprotein.     

The N-terminus of vaccinia virus host range protein C7L is essential for function

Vaccinia virus (VACV), a member of the Poxviridae family of large double-stranded DNA viruses, is being used as a smallpox vaccine as well as an expression vector for immunization against other infectious diseases and cancer. The host range of wild type VACV is very broad among mammalian cells. C7L is a host range gene identified in VACV and is well conserved in mammalian poxviruses except for parapoxviruses and molluscum contagiosum virus. The molecular mechanisms by which the C7L gene exerts host range function are not well understood. The C7L protein does not have any known conserved domains or show sequence similarity to cellular proteins or viral proteins other than the C7L homologs in mammalian poxviruses. We generated recombinant vaccinia viruses carrying deletion mutants of the C7L gene using NYVAC as a parental strain and found that the N-terminus is essential for host range function of C7L, which is consistent with a previous report that showed that homology among C7L homologs are greater near the N-terminus than the C-terminus.     

乙型肝炎病毒X蛋白抑制SFRP5启动子的活性

 目的:探讨乙型肝炎病毒X蛋白(HBx)抑制分泌型卷曲相关蛋白5(SFRP5)启动子活性的分子机制。方法:扩增不同长度的SFRP5启动子区片段,构建到萤光素酶报告基因载体pGL3-Basic上,并将构建好的重组质粒转染入正常肝细胞系LO2细胞,再分别感染编码HBx的腺病毒（Ad-HBx）或编码绿色荧光蛋白的腺病毒（Ad-GFP）,荧光显微镜下观察病毒感染效率,检测萤光素酶活性以观察HBx对SFRP5启动子活性的影响。结果:(1) 含不同长度的SFRP5启动子区截短突变报告质粒构建成功。(2) 截短突变报告质粒的萤光素酶活性与pGL3-Basic对照组相比,分别为(6.32±0.04)倍(-478 bp~+47 bp)、(5.79±0.32)倍(-811 bp~+47 bp)、(3.59±0.34)倍(-1 235 bp~+47 bp)、(3.86±0.39)倍(-1 677 bp~+47 bp)、(3.26±0.42)倍(-2 072 bp~+47 bp) (均P<0.05)。过表达HBx可以明显抑制SFRP5启动子活性,抑制率分别为44%(-478 bp~+47 bp) (P<0.05)、 46%(-811 bp~+47 bp) (P<0.05)、 28%(-1 235 bp~+47 bp)、 24%(-1 677 bp~+47 bp)和40%(-2 072 bp~+47 bp)。结论: HBx可以明显抑制SFRP5启动子区活性,可能导致SFRP5基因表达下调。     

The polybasic region is not essential for membrane binding of the matrix protein M1 of influenza virus

The matrix protein M1, the organizer of assembly of influenza virus, interacts with other virus components and with cellular membranes. It has been proposed that M1 binding to lipids is mediated by its polybasic region, but this could hitherto not been investigated in vivo since M1 accumulates in the nucleus of transfected cells. We have equipped M1 with nuclear export signals and showed that the constructs are bound to cellular membranes. Exchange of the complete polybasic region and of further hydrophobic amino acids in its vicinity did not prevent association of M1 with membranes. We therefore suppose that M1 probably interacts with membranes via multiple binding sites.