2''-5''寡腺苷酸合成酶1基因多态性与乙型肝炎病毒易感性和抗病毒疗效的相关性

人体感染HBV后,可自身清除病毒或持续性感染,在成人感染者中有5%～10%不能清除病毒,导致慢性病毒携带状态。其造成肝损伤的程度不一,发展为肝硬化或肝癌进程不一、药物治疗的反应个体差异亦很大,宿主的遗传因素对乙型肝炎的这些临床转归起了关键作用。目前干扰素诱导抗病毒基因2′-5′寡腺苷酸合成酶1（OAS1）与HBV易感性和抗病毒疗效的相关性研究鲜见报道，[第一段]     

Molecular cloning and sequence of partial cDNA for interferon-induced (2''-5'')oligo(A) synthetase mRNA from human cells.

By using a translation assay in oocytes, a 17S RNA fraction coding for the interferon-induced (2'-5')oligo(A) synthetase was purified from human cells. A cDNA library was prepared by cloning in Escherichia coli plasmid pBR322 and screened by positive hybridization-translation in oocytes. A cDNA clone corresponding to the (2'-5')oligo(A) synthetase mRNA was identified. In SV80 cells, this E cDNA recognizes three RNAs of 1.65, 1.85, and 3.6 kilobases, which are present only after interferon treatment of the cells. In Namalva cells, mainly one RNA of 1.8 kilobases is seen.     

Loss of (2''-5'')oligoadenylate synthetase activity by production of antisense RNA results in lack of protection by interferon from viral infections.

An expression vector was constructed that carries part of the human BK papovavirus with 0.5 kilobases of (2'-5')oligoadenylate (2-5A) synthetase cDNA inserted in inverted orientation downstream from the virion proteins (VP) promoter and the neomycin-resistance gene neo under the control of a simian virus 40 promoter. Cells transfected with this vector and selected for resistance to the neomycin derivative G418 synthesized RNA complementary to 2-5A synthetase mRNA. These cells lacked 2-5A synthetase activity, and the enzyme was not inducible by interferon. In contrast, 2-5A synthetase was induced in cells transfected with a control vector without the cDNA insert. Such cells were protected by interferon from RNA viruses, whereas cells lacking 2-5A synthetase were not protected from encephalomyocarditis virus, vesicular stomatitis virus, and Sindbis virus but were fully protected from influenza virus. These findings show that a high level of 2-5A synthetase is required for interferon-induced protection from the cytoplasmic RNA viruses tested.     

Absence of the 40-kDa form of (2''-5'')oligoadenylate synthetase and its corresponding mRNA from skin fibroblasts derived from Alzheimer disease patients.

Cultured skin fibroblasts derived from Alzheimer disease patients fail to express the 1.6-kilobase (kb) mRNA and the corresponding 40-kDa form of (2'-5')oligoadenylate (2-5A) synthetase when exposed to interferon. In addition, the 3.6-kb mRNA, which is present in normal fibroblasts, is barely detectable in the Alzheimer disease counterpart. The deficiency of the 2-5A synthetase 1.6-kb mRNA and its corresponding protein is not related to an impairment of interferon receptors but most probably represents an alteration in the expression of the 2-5A synthetase gene. The data have potential implications for the diagnosis of Alzheimer disease and demonstrate that the absence of a specific form of 2-5A synthetase is linked to a disease state.     

Signal transduction pathways in the induction of 2'',5''-oligoadenylate synthetase gene expression by interferon alpha/beta.

Treatment of quiescent BALB/c mouse 3T3 cells with murine interferon alpha/beta (IFN-alpha/beta) (1000 units/ml) leads to the appearance at 4 hr of 1.7-kilobase 2',5'-oligoadenylate (2',5'-OAS)mRNA as detected by Northern blot analysis. This mRNA accumulates for at least 18 hr. Two protein kinase C activators, 1,2-dioctanoyl glycerol and phorbol 12-myristate 13-acetate, suppress, whereas the calcium ionophore ionomycin enhances, the IFN-alpha/beta-induced expression of 2',5'-OAS mRNA. The 8-bromo and dibutyryl analogs of cAMP and the adenylate cyclase activator forskolin did not affect the induction of 2',5'-OAS mRNA by IFN-alpha/beta. In the absence of IFN-alpha/beta, the above agents used either singly or in combinations, did not induce 2',5'-OAS mRNA expression nor did platelet-derived growth factor (1-2 units/ml), fibroblast growth factor (6-100 ng/ml), or bovine serum (10-20%). Bovine serum also did not affect 2',5'-OAS mRNA induction by IFN-alpha/beta. The poly(ADP)-ribose synthetase inhibitor 3-aminobenzamide suppressed IFN-alpha/beta-induced 2',5'-OAS gene expression. These results suggest that in quiescent BALB/c 3T3 cells (i) the 2',5'-OAS gene is not responsive to the three major signal transduction pathways activated by diacylglycerol, Ca2+, and cAMP; (ii) induction of the 2',5'-OAS gene by IFN-alpha/beta is decreased by activation of the protein kinase C pathway but enhanced by elevation of intracellular [Ca2+].     

Core (2''-5'')oligoadenylate and the cordycepin analog: inhibitors of Epstein--Barr virus-induced transformation of human lymphocytes in the absence of interferon.

The 3'-deoxyadenosine (cordycepin) analog of (2'-5')oligo(A) [(2'-5')oligoadenylate with a triphosphate at the 5' end], synthesized enzymatically from cordycepin 5'-triphosphate in lysed rabbit reticulocytes or L-cell extracts was (i) inhibitory to translation in lysed rabbit reticulocytes and (ii) metabolically stable in extracts of either L cells or C85-5C lymphoblasts. The 5' dephosphorylated (core) (2'-5')oligo(A) and the core cordycepin analog can replace human fibroblast interferon in preventing the transformation of human lymphocytes after infection with Epstein--Barr virus B95-8 (EBV) as determined by the decreased incorporation of [3H]thymidine into cellular DNA and the inhibition of morphological transformation of EBV-infected lymphocytes. Whereas the naturally occurring core (2'-5')oligo(A) was cytotoxic to uninfected lymphocytes and proliferating lymphoblasts, the core cordycepin analog was not. Human leukocyte interferon was more effective than human fibroblast interferon in the inhibition of EBV-induced transformation of human umbilical cord lymphocytes and adult peripheral blood lymphocytes.     

Molecular cloning of the cDNA encoding the Epstein-Barr virus/C3d receptor (complement receptor type 2) of human B lymphocytes.

Complementary DNA clones for complement receptor type 2 (CR2), the B-lymphocyte membrane protein that serves as the receptor for Epstein-Barr virus and the C3d complement fragment, were obtained by screening a lambda gt11 library generated from Raji B lymphoblastoid cell mRNA. A 4.2-kilobase (kb) clone, representing the entire coding sequence of the protein plus untranslated 5' and 3' nucleotide sequences was obtained and sequenced. The 4.2-kb clone, which contains all but about 500 base pairs (bp) of the 5' untranslated region of the full-length CR2 mRNA, consists of 63 bp of 5' untranslated nucleotide sequence followed successively by a start codon, a 20-amino acid hydrophobic signal peptide, 1005 amino acids having a repeating motif, a 28-amino acid probable transmembrane domain, and a 34-amino acid cytoplasmic tail. The deduced amino acid sequence of the protein indicates that the extracellular domain consists entirely of 16 tandemly arranged repeating elements, each 60-75 amino acids in length, which are identified by multiple conserved residues. This repeating motif also occurs in the C3b/C4b receptor, several complement proteins, and a number of noncomplement proteins. In CR2, the 16 repeats occur in four clusters of four repeats each. Approximately 10% of the deduced amino acid sequence, including the amino and carboxyl termini, was confirmed by amino acid sequencing of tryptic peptides derived from purified CR2. The nucleotide and derived amino acid sequence of CR2 and related studies are presented here.     

Randomised controlled trial of interferon alfa 2A (rbe) (Roferon-A) for the treatment of chronic hepatitis B virus (HBV) infection: factors that influence response.

In a randomised controlled trial recombinant interferon alpha 2A (Roferon-A, rIFN alfa A) given at a dosage of 10 million units (MU)/m2 thrice weekly for six months was significantly better (p less than 0.02) than no treatment in producing a sustained loss of hepatitis Be antigen (HBeAg) in hepatitis B virus (HBV) chronic carriers. Although lower doses (5 MU/m2 and 2.5 MU/m2) also produced some responses, the seroconversion rate was not significantly greater than that observed in the control group. Sixteen of the 45 patients receiving interferon were human immunodeficiency virus (HIV) antibody positive: none of these responded. Forty one per cent of the anti-HIV negative patients receiving interferon (12/29, p less than 0.005) lost HBeAg and 17% (5/29) lost hepatitis B surface antigen (HBsAg). The response rate among these anti-HIV negative patients receiving at least three months therapy was 46% and 19% respectively. Low pretreatment HBV-DNA and absence of anti-HIV were the only significant independent variables predicting response to therapy (p less than 0.03 and p less than 0.05 respectively). In six patients, neutralising antibodies to alpha interferon were detected during therapy, the majority being non-responders.     

Reconstitution of functional receptors for human granulocyte/macrophage colony-stimulating factor (GM-CSF): evidence that the protein encoded by the AIC2B cDNA is a subunit of the murine GM-CSF receptor.

The high-affinity receptor for human granulocyte/macrophage colony-stimulating factor (hGM-CSF) is composed of two subunits, alpha and beta. The alpha subunit binds GM-CSF with low affinity, whereas the beta subunit does not bind GM-CSF by itself. The alpha and beta subunits together form the high-affinity GM-CSF receptor. The beta subunit has extensive sequence homology with the mouse interleukin 3 (IL-3) receptor (AIC2A) and its homologue (AIC2B) that does not bind IL-3 or other cytokines including GM-CSF. To examine the function of these receptor components, we expressed the alpha subunit of the hGM-CSF receptor with the human beta subunit or the mouse AIC2A or AIC2B in a mouse IL-3-dependent pro-B-cell line, Ba/F3, and in a mouse IL-2-dependent T-cell line, CTLL2. Coexpression of the alpha and beta subunits in Ba/F3 and CTLL2 cells resulted in high-affinity hGM-CSF binding and growth response to low concentrations of hGM-CSF. Whereas Ba/F3 cells expressing the alpha subunit alone proliferated in response to high concentrations of hGM-CSF, CTLL2 cells expressing the alpha subunit alone did not respond to hGM-CSF at all. Since Ba/F3 cells express endogenous AIC2A and AIC2B whereas CTLL2 expresses neither of them, we examined the possibility that either AIC2A or AIC2B is involved in the formation of a functional GM-CSF receptor. The expression of the human alpha subunit with AIC2B, but not with AIC2A, in CTLL2 cells conferred a growth response to hGM-CSF. These results indicate that the beta subunit of the GM-CSF receptor is required for generation of growth signals and that AIC2B is likely the beta subunit of the mouse GM-CSF receptor.