Expression of the interferon-inducible proteins MxA and IFI16 in liver allografts

Aims:  To test the hypothesis that the activation of the interferon (IFN) system pathways might link hepatitis C virus (HCV) recurrence in the liver allograft with acute cellular rejection.
Methods and results:  In this retrospective study, allograft biopsy specimens from 28 adult patients (14 HCV+ and 14 HCV−) who had undergone their first liver transplantation were analysed. Eleven biopsy specimens showed acute cellular rejection (Banff rejection activity index score ≥3). Specimens were immunostained for two IFN-inducible proteins, MxA and IFI16, and for CD45. The predominant MxA reactivity pattern was hepatocytic, whereas IFI16 was expressed in both the hepatocellular and inflammatory compartments. Moderate to strong MxA expression in hepatocytes was associated positively with rejection score ( P  < 0.01), donor's age ≤45 years ( P  < 0.05) and aspartate aminotransferase levels >40 U/l on the day of biopsy ( P  < 0.05), and inversely with infiltration of portal triads by IFI16+/CD45+ cells ( P  < 0.005) and time to progression beyond Ishak stage 2 of recurrent hepatitis C ( P  < 0.01). On multivariate analysis, MxA expression in hepatocytes was independently associated with allograft rejection and donor's age.
Conclusions:  Acute allograft rejection and recurrence of HCV infection in the liver allograft appear to intersect in the IFN system pathways.     

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.     

人B7H4启动子荧光素酶报告基因载体的构建及活性分析

为构建人B7H4基因启动子荧光素酶报告基因载体,以人外周血单个核细胞基因组DNA为模板,PCR扩增3条不同长度人B7H4启动子序列,并插入PGL3-Basic荧光素酶报告基因载体中;待测序验证后,将3个重组质粒及pRL-TK内参质粒分别共转染HEK-293T细胞,采用双荧光素酶报告基因系统检测其启动子活性。测序结果显示构建的3个人B7H4基因启动子重组载体序列正确;重组载体转染HEK-293T细胞,经双荧光素酶报告基因检测确定重组载体有启动子活性,其中PGL3-hB7H4-0.5kb重组载体的转录活性较高。本研究成功构建了3条含不同长度的B7H4启动子序列的荧光素酶报告基因系统,为后续分析人B7H4启动子的转录调控元件及肿瘤微环境中调控B7H4表达的作用因素等研究奠定了实验基础。     

重型肝炎相关的人纤维介素基因启动子的克隆及功能分析

目的明确在病毒蛋白HBc和HBx作用下,hfgl2基因5′端非编码区对转录激活起重要作用的转录调控序列。方法运用基因重组的方法,构建一系列5′端缺失而保留共同3′端的hfgl2基因启动子虫荧光素酶报告质粒,将其分别与病毒蛋白HBc和HBx真核表达质粒共转染CHO细胞和HepG2细胞,检测各组细胞的相对荧光素酶活性。结果酶切鉴定以及DNA测序等证实一系列hfgl2基因启动子虫荧光素酶报告基因质粒构建成功,系列启动子缺失试验证实:在hfgl2基因启动子-817位至-467位(相对于转录起始点)之间存在着激活该基因的调控序列。结论在HBV病毒蛋白HBc及HBx作用下,hfgt2基因的启动子区存在一个与其转录表达有关的调控序列,探讨了重型肝炎相关的hfgl2基因高度表达的分子机制,为下一步研究相关的顺式作用元件及反式作用因子奠定了基础。     

用双向凝胶电泳-飞行时间质谱技术分析HBV感染肝组织蛋白质组

目的 比较正常肝组织与HBV感染肝组织中蛋白质表达谱的改变,筛查在HBV致病过程中相关蛋白质分子表达的不同.方法 应用双向聚丙烯酰胺凝胶电泳,对3例正常肝组织(A组),3例HBV感染肝组织(B组,均为组织HBsAg阳性,外周血HBsAg阳性、抗-Hbe阳性、抗-HBc阳性)的蛋白质组表达谱进行差异分析,应用基质辅助激光解吸离子化飞行时间质谱仪对差异蛋白质点进行了鉴定.结果 相同条件下对各组总蛋白样品分别进行3次双向电泳,各组检测到的蛋白点数如下:A组(1125±56)个,B组(1203±42)个.以各组中2-DE效果好、点数多的一块胶作为参考胶,分别进行2组间的匹配分析,筛选出两组之间差异大于或等于2倍的蛋白点40个.应用质谱鉴定出人线粒体醛脱氢酶H链、结合珠蛋白Hp2、抗过氧化物蛋白2等22种蛋白质.结论 HBV感染所致慢性炎症能使肝组织表现出明显的差异性蛋白表达谱.     

Computer predictions of functional,topogenic, and antigenic domains in human immunodeficiency virus-2 envelope glycoprotein

The gp160 of HIV-2 was studied with the aid of computer programs that provide the hydrophilicity, surface probability, flexibility, and antigenicity index of the amino-acid sequence in a polypeptide chain. Such analyses allow the identification of hydrophobic amino-acid domains in the polypeptide chain that may serve as putative proteolytic cleavage signals and putative antigenic domains. It was possible to define the function of hydrophobic domains in the polypeptide chain that serve as signals and amino-acid sequences involved in the transfer of the polypeptide through the cellular membrane by the cellular signal recognition protein (SRP) complex. By comparison to reported properties of HIV-1 gp160 and SIV_MAC gp160, it was possible to define antigenic domains in the loops of gp120 resulting from the reported interchain disulfide bonds defining putative antigenic domains specific for HIV-2.     

Inhibition of influenza C viruses by human MxA protein

Human MxA protein was analyzed for its ability to inhibit the replication of different influenza C viruses. Three laboratory derivatives of viral strain C/Ann Arbor/1/50 were investigated, namely the parental wild-type virus C/AA-wt, the persistent variant C/AA-pi and the highly cytopathogenic variant C/AA-cyt. In addition, strain C/Paris/214/91 isolated from an influenza patient was used. Multiplication of all four viruses was suppressed in MxA-expressing Vero cells, as indicated by a decrease in viral RNA synthesis, viral protein synthesis, virion production and induction of a cytopathic effect. Inhibition correlated with the level of MxA expression. Furthermore, inhibition was independent of cell clone-specific differences in expression of virus receptors, as demonstrated by receptor reconstitution experiments. Thus, human MxA protein has antiviral activity against influenza C viruses.     

Inhibition of Dugbe nairovirus replication by human MxA protein

Sensitivity to the interferon-induced protein, MxA, has previously been demonstrated for viruses belonging to the Orthobunyavirus, Hantavirus and Phlebovirus genera of the Bunyaviridae family. We have extended these findings to a member of the fourth and remaining genus containing viruses that infect man and other animals, the nairovirus Dugbe virus (DUGV). Indirect immunofluorescence experiments using VA9 cells (Vero cells permanently transfected with MxA cDNA) revealed strongly reduced DUGV antigen expression, suggesting that MxA inhibited DUGV replication. Western and Northern blot analyses showed significantly lower DUGV nucleocapsid (N) protein expression and DUGV genomic RNA, respectively, in the presence of MxA. Viral titres were also reduced by more than two orders of magnitude in VA9 cells compared with control VN36 cells. This finding may have application to nairovirus therapeutics.     

Molecular cloning of a macrophage-derived, interferon-inducible secreted immunoglobulin-binding protein.

We describe the molecular cloning of a 1803-bp cDNA coding for a product termed interferon-induced immunoglobulin-binding protein (IIBP) from a library of IFN-alpha-induced primary bone marrow macrophages. The open reading frame encodes a protein of 26-kDa containing two immunoglobulin-like and one Fc receptor-like domain. Due to the constitutive release of low amounts of IFN-beta, the IIBP mRNA is already present in macrophage-colony-stimulating factor-cultured macrophages. Its expression could be blocked in the presence of either anti-IFN-beta or interleukin-4, which down-regulates the endogenous IFN-beta production. Upon addition of rIFN-alpha4 a 3-5-fold superinduction of IIBP mRNA was observed. Rat monoclonal antibodies detected a protein of the predicted size exclusively in cell culture supernatants of primary bone marrow macrophages and a B-cell line. In immunoprecipitation experiments an unknown 30-kDa protein co-precipitated. The secreted IIBP showed considerable binding to nonspecific rat IgG2a and could be precipitated using mouse IgG2a, IgG2b and IgG3 antibodies of irrelevant specificities, indicating that this gene product is a novel secreted immunoglobulin-binding protein with a new IgG isotype binding pattern that differs to that of known Fc receptors.