Human hepatitis B virus X protein augments the DNA binding of nuclear factor for IL-6 through its basic-leucine zipper domain

The X gene product of human hepatitis B virus, HBx, transactivates the expression of viral and cellular genes through a wide variety of cis elements, including the nuclear factor for IL-6 (NF-IL6) binding sites, although HBx does not appear to bind DNA directly. We previously reported that HBx transactivated the interleukin 8 promoter through NF-kappaB binding site and C/EBP-like binding site (NF-IL6 binding site). In this study, the interactions were examined between NF-IL6 and HBx using recombinant proteins. In a DNA-protein binding assay, the formation of a specific complex between NF-IL6 and a DNA probe harboring an NF-IL6 binding site was increased by the addition of either the full or the C-terminal 104 amino acids of HBx. A direct protein-protein binding assay (far-Western blot) revealed the direct interaction between the C-terminal 104 amino acids of HBx and the basic region-leucine zipper domain of NF-IL6. These results indicate that HBx alters the DNA-binding affinity of NF-IL6 through the direct interaction between the C-terminal domain of HBx and the basic region-leucine zipper domain of NF-IL6.     

Mutational analysis of enhancer domains responsive to trans-activation by the X gene of human hepatitis B virus

Summary The X gene product of human hepatitis B virus (HBV) trans-activates the HBV enhancer. In order to identify domains responsive to trans-activation by the X gene, we introduced a series of mutations into the HBV enhancer and assayed the enhancer activities in the presence of the X gene product. Our results suggest that the EP domain of the enhancer is essential for trans-activation by the X gene.     

Cooperative transformation of murine fibroblast NIH3T3 cells by hepatitis C virus core protein and hepatitis B virus X protein

Co-infection with hepatitis B virus (HBV) and hepatitis C Virus (HCV) is associated with increased frequency in the development of hepatocellular carcinoma (HCC). Here, we demonstrated that HBV X protein (HBx) and HCV core cooperate to transform mouse fibroblast NIH3T3 cells. They additively stimulated cell growth, especially in the absence of serum growth factors. In addition, co-expression of HBx and HCV core had additive effects on the induction of anchorage-independent cell growth as well as on the secretion of matrix metalloproteases, which may contribute to increased metastatic potential. Furthermore, the cells expressing both viral proteins exhibited higher tumorigenicity, as demonstrated in athymic nude mice.     

慢性乙肝患儿HBV基因型与乙肝病毒大蛋白关系探讨

目的 探讨慢性乙型肝炎患儿HBV基因型与乙肝病毒大蛋白的关系.方法 采用实时荧光PCR法和ELISA法分别检测138例处于乙肝病毒活动期的慢性乙型肝炎患儿血清中的HBV DNA和乙肝病毒大蛋白并鉴定其基因型.结果 乙肝病毒大蛋白吸光度与HBV DNA载量存在正相关（r=0.85,P＜0.05）;HBV基因B型与HBV基因C型的ALT水平、乙肝病毒大蛋白吸光度和HBVDNA载量差异无统计学意义（P＞0.05,P＞0.05,P＞0.05）.结论 乙肝病毒大蛋白水平与HBVDNA载量具有良好的正相关性,表明乙肝病毒活动期的慢性乙肝患者体内乙肝病毒大蛋白与病毒复制程度密切相关,乙肝病毒基因型与乙肝病毒大蛋白无关.     

A human-derived protein SBP (HBsAg-binding protein) can bind to hepatitis B virus surface antigen (HBsAg) and enhance the immune response to hepatitis B virus (HBV) vaccine

A high titer of antibody to HBsAg (Hepatitis B virus surface antigen) (anti-HBs) is a requisite for the prevention of HB (Hepatitis B), and adjuvants generally play a great role in eliciting special anti-HBs to HB vaccine. However, adjuvants still need to be improved because of their shortages such as unremarkable efficacy, undesirable side effect or poor security. In this study, we used HBsAg separated from HB patient sera to screen a human liver cDNA expression library, and found a novel HBsAg-binding protein (SBP), which is located at the human chromosome 14q32.33 and is similar to human IgG heavy chain in structure. Western blot demonstrated that SBP existed in both healthy human sera and HB patient sera. Furthermore, SBP could bind to HBsAg by its N-terminal domain. Notably, we confirmed that SBP could promote dendritic cells (DC) to phagocytize HBsAg more effectively and enhance the immunogenicity of HB vaccine, when SBP was mixed proportionally with HBsAg and the resulting mixture was infused into mice. These results suggest that SBP could be developed into a safe and promising adjuvant of HB vaccine.     

The NH2-terminal domain of rat CD2 binds rat CD48 with a low affinity and binding does not require glycosylation of CD2

CD2, CD48 and CD58 are structurally similar cell adhesion-molecules forming a subset of the immunoglobulin superfamily (IgSF). In humans CD58 is a ligand for CD2 while in mice CD2 binds CD48. We constructed a soluble chimeric molecule comprising the extracellular portion of rat CD48 and domains 3 and 4 of rat CD4 (sCD48-CD4) and used it to examine whether CD2 is a ligand for CD48 in rats. sCD48-CD4-coated polystyrene Dynabeads? formed rosettes on rat CD2-transfected COS-7 cells, and this rosetting was blocked by anti-CD2 (OX34) and anti-CD48 (OX45) monoclonal antibodies. We used sucrose-gradient ultracentri-fugation to show that sCD48-CD4 binds, in solution, to soluble forms of rat CD2 including the single NH₂-terminal IgSF domain of rat CD2 expressed in bacteria. The upper limit of the affinity of the rat CD48-CD2 interaction is 4 × 10⁵ M^?1, lower than the published affinity of human CD2 for CD58. These results show that rat CD48 binds CD2 on its NH₂-terminal IgSF domain with a low affinity and that binding is independent of glycosylation.     

Huntingtin-associated protein 1 (HAP1) binds to a Trio-like polypeptide, with a rac1 guanine nucleotide exchange factor domain

Huntington's disease (HD) occurs when the widely expressed protein huntingtin contains an expanded glutamine repeat. The selective degeneration and neuronal morphologic abnormalities of HD may involve interactions with proteins that bind to huntingtin, such as HAP1. The biological significance of this interaction is unclear because neither HAP1 nor huntingtin have significant homology to known proteins. Therefore, we sought to identify HAP1-binding proteins. Using the yeast two-hybrid system, we isolated a rat cDNA encoding part of a protein that interacts with HAP1, and we confirmed the specificity of this interaction using an in vitro protein-binding assay. We called the protein Duo because it is closely related to the human protein Trio but is shorter. Northern blot analysis indicates brain-specific expression of Duo. Human Duo contains a guanine nucleotide exchange factor (GEF) domain that is likely to be rac1-specific, a pleckstrin homology (PH) domain and spectrin-like repeat units. These data support the hypothesis that huntingtin is involved in vesicle trafficking and cytoskeletal functions, and raise the possibility of a role for huntingtin in the regulation of a ras-related signaling pathway.      

Epitope mapping of the PreS1 domain of the hepatitis B virus large surface protein.

The large (L) surface glycoprotein of hepatitis B virus is an important component of the virion envelope derived from translation initiation at the 5' end of the PreS1 domain of the surface antigen open reading frame. Since key roles in virion assembly and infectivity have been postulated for this protein, further understanding of its structure and topology is important. To this end we have mapped the epitopes recognized by a panel of monoclonal antibodies specific for this polypeptide by examining their reactivity with a series of deletion mutants of the PreS1 region expressed in cultured cells. On the basis of this and other techniques, the antibodies fall into two groups mapping to two distinct epitopes spanning residues 27-35 and 72-78, respectively. Immunoprecipitation studies indicate that both regions are exposed on the surface of HBV-encoded particles.     

小鼠多囊蛋白1氨基端多克隆抗体的制备和其生物特性分析

目的 制备针对多囊蛋白1(PC1)胞外区的抗体,为分析PC1胞外区生物特性和功能提供工具.方法 根据生物信息学分析结果,PCR扩增编码小鼠Pkd1的氨基端474E-640L的eDNA片段.将该片段克隆到GST融合蛋白表达载体上,在IPTG诱导下产生小鼠Pkdl抗原(mPkd1-N).纯化浓缩目的蛋白并制备兔抗PC1氨基端多克隆抗体(mPkd1-Np),并以Western blot法、免疫组化以及免疫荧光方法分析该兔抗PC1氨基端抗体的特异性.结果 成功地构建了mPkd1-N片段真核及原核表达载体,在大肠杆菌中实现表达,并制备了抗小鼠PC1氨基端的多克隆抗体mPkd1-Np.通过生化和细胞学方法证实了该抗体针对PC1的特异性.结论 成功制备了高效价、特异性的兔抗mPkdl-Np多克隆抗体.     

Identification of human hepatocyte protein(s), which binds specifically to the recombinant envelope-2/non-structural-1 protein of hepatitis C virus

Hepatitis C virus (HCV), which is the major pathogen responsible for human chronic liver disease, has special tropism for hepatocytes. Although, low-density lipoprotein receptor, CD81 and negatively charged glycosaminoglycans have been proposed as candidate receptors for HCV, no confirmed receptor(s) on the hepatocytes have been identified to date. It is also suggested that additional, yet unidentified, cellular proteins may be involved in the host-viral interaction. Therefore, this study was conducted with the main aim to identify hepatocyte protein(s) that may have affinity for the HCV structural protein, envelope-2/non-structural-1 (E2/NS1) protein. For the binding studies, hepatocytes were isolated from fresh normal human liver tissues. The hepatocyte proteins on the nitrocellulose paper were reacted with recombinant E2/NS1 protein and anti-E2 (rabbit). In another approach, to rule out the possibility of binding of rec-E2/NS1 with the hepatocyte cytoplasmic proteins, hepatocyte plasma membrane proteins were passed through CNBr-activated and recombinant E2/NS1 bound sepharose-4B column. The recombinant E2/NS1 binding hepatocyte plasma membrane protein(s) were eluted and were then analyzed. Altogether, our data suggest that E2/NS1 protein of HCV binds to two hepatocyte proteins of molecular weights 25-28 kDa and 59-60 kDa. These results indicate the possible role of the above proteins (25-28 kDa and 59-60 kDa) in the viral binding to the hepatocytes.