High levels of unintegrated HIV-1 DNA in brain tissue of AIDS dementia patients

In the host cell, retroviral DNAs exist in three main forms: unintegrated linear, unintegrated circular, and integrated (the provirus). High levels of unintegrated forms of retroviral DNA often correlate with superinfection and accompanying cytopathic effects, as, for example, in the case of feline acquired immunodeficiency. In culture, HIV-1 infection also results in high levels of unintegrated viral DNA although direct correlations with cytopathicity have not been made. The low frequency of HIV-1-infected cells in patients has made it difficult to determine the structure of the viral DNA in fresh tissue samples from AIDS patients by standard methods such as Southern hybridization. The PCR technique however, which allows the detection of viral DNA at levels far below that possible by other hybridization methods is, in its conventional form, of limited use for quantitative analysis. To study the amount and form of HIV-1 DNA in primary tissue of AIDS patients we have therefore modified the PCR method. Our results indicate that each of the three species of viral DNA are detectable in blood and brain of AIDS patients, and that in autopsy samples from patients with HIV encephalitis there is a considerably higher proportion of unintegrated viral DNA.     

水溶性C60-脱氢枞胺衍生物的合成及生物活性

以C60-脱氢枞胺吡咯烷衍生物和C60-脱氢枞胺氮烯加成物为原料,通过两种不同的水性化途径分别合成了两种水溶性的C60-脱氢枞胺富勒醇衍生物和两种水溶性的C60-脱氢枞胺甘氨酸衍生物。产物经TEM、FT-IR、ESI-MS、1H NMR和HPLC等分析方法检测确认为目标产物,且为加成度不同的混合物。对合成出的4种水溶性C60-脱氢枞胺衍生物进行了生物活性测试。结果表明,4种水溶性C60-脱氢枞胺衍生物对rTaq DNA聚合酶和HIV-1反转录酶均表现出一定的抑制作用,对卵巢癌细胞株几乎不表现出抑制活性。     

Molecular architecture of native HIV-1 gp120 trimers

The envelope glycoproteins (Env) of human and simian immunodeficiency viruses (HIV and SIV, respectively) mediate virus binding to the cell surface receptor CD4 on target cells to initiate infection. Env is a heterodimer of a transmembrane glycoprotein (gp41) and a surface glycoprotein (gp120), and forms trimers on the surface of the viral membrane. Using cryo-electron tomography combined with three-dimensional image classification and averaging, we report the three-dimensional structures of trimeric Env displayed on native HIV-1 in the unliganded state, in complex with the broadly neutralizing antibody b12 and in a ternary complex with CD4 and the 17b antibody. By fitting the known crystal structures of the monomeric gp120 core in the b12- and CD4/17b-bound conformations into the density maps derived by electron tomography, we derive molecular models for the native HIV-1 gp120 trimer in unliganded and CD4-bound states. We demonstrate that CD4 binding results in a major reorganization of the Env trimer, causing an outward rotation and displacement of each gp120 monomer. This appears to be coupled with a rearrangement of the gp41 region along the central axis of the trimer, leading to closer contact between the viral and target cell membranes. Our findings elucidate the structure and conformational changes of trimeric HIV-1 gp120 relevant to antibody neutralization and attachment to target cells.     

Crystal structure of the anti-viral APOBEC3G catalytic domain and functional implications

The APOBEC family members are involved in diverse biological functions. APOBEC3G restricts the replication of human immunodeficiency virus (HIV), hepatitis B virus and retroelements by cytidine deamination on single-stranded DNA or by RNA binding. Here we report the high-resolution crystal structure of the carboxy-terminal deaminase domain of APOBEC3G (APOBEC3G-CD2) purified from Escherichia coli. The APOBEC3G-CD2 structure has a five-stranded beta-sheet core that is common to all known deaminase structures and closely resembles the structure of another APOBEC protein, APOBEC2 (ref. 5). A comparison of APOBEC3G-CD2 with other deaminase structures shows a structural conservation of the active-site loops that are directly involved in substrate binding. In the X-ray structure, these APOBEC3G active-site loops form a continuous 'substrate groove' around the active centre. The orientation of this putative substrate groove differs markedly (by 90 degrees) from the groove predicted by the NMR structure. We have introduced mutations around the groove, and have identified residues involved in substrate specificity, single-stranded DNA binding and deaminase activity. These results provide a basis for understanding the underlying mechanisms of substrate specificity for the APOBEC family.     

尿激酶体外抑制人体免疫缺损病毒的侵染能力

人体免疫缺损病毒的包膜蛋白gp120的V3环区包含一段在人类蛋白质中很少出现的高度保守序列，但这段序列与纤溶酶原被纤溶酶原激活剂酶切位点附近序列有同源性．由于V3环区在人体免疫缺损病毒侵染细胞过程中的重要性，评估了尿激酶对人体免疫缺损病毒侵染能力的影响．通过检测逆转录酶活力，P24抗原的表达和合胞体形成情况发现尿激酶可以抑制人体免疫缺损病毒对多种淋巴瘤和白血病细胞系，如MT4、CCM、H9和外周血单核细胞的侵染能力，并且这种抑制与尿激酶浓度呈剂量依赖关系．那些能够被尿激酶抑制的人体免疫缺损病毒株其V3环区序列必须与纤溶酶原激活区亭列同源，实验事常用病毒株包括BRU和RF以及某些野生病毒株．研究结果显示尿激酶在体外实验中可以抑制人体免疫缺损病毒的侵染能力．     

Structure of DNase I at 2.0 A resolution suggests a mechanism for binding to and cutting DNA

Bovine pancreatic deoxyribonuclease I (DNase I), an endonuclease that degrades double-stranded DNA in a nonspecific but sequence-dependent manner, has been used as a biochemical tool in various reactions, in particular as a probe for the structure of chromatin and for the helical periodicity of DNA on the nucleosome and in solution. Limited digestion by DNase I, termed DNase I 'footprinting', is routinely used to detect protected regions in DNA-protein complexes. Recently, we have solved the three-dimensional structure of this glycoprotein (relative molecular mass 30,400) by X-ray structure analysis at 2.5 A resolution and have subsequently refined it crystallographically at 2.0 A. Based on the refined structure and the binding of Ca2+-thymidine 3',5'-diphosphate (Ca-pTp) at the active site, we propose a mechanism of action and present a model for the interaction of DNase I with double-stranded DNA that involves the binding of an exposed loop region in the minor groove of B-DNA and electrostatic interactions of phosphates from both strands with arginine and lysine residues on either side of this loop. We explain DNase I cleavage patterns in terms of this model and discuss the consequences of the extended DNase I-DNA contact region for the interpretation of DNase I footprinting results.     

Structure refined to 2A of a nicked DNA octanucleotide complex with DNase I

The cutting rates of bovine pancreatic deoxyribonuclease I (DNase I) vary along a given DNA sequence, indicating that the enzyme recognizes sequence-dependent structural variations of the DNA double-helix. In an attempt to define the helical parameters determining this sequence-dependence, we have co-crystallized a complex of DNase I with a self-complementary octanucleotide and refined the crystal structure at 2 A resolution. This structure confirms the basic features of an early model, namely that an exposed loop of DNase I binds in the minor groove of B-type DNA and that interactions do occur with the backbone of both strands. Nicked octamer duplexes that have lost a dinucleotide from the 3'-end of one strand are hydrogen-bonded across a two-fold axis in the crystal to form a quasi-continuous double helix of 14 base pairs. The DNA 14-mer has a B-type conformation and shows substantial distortion of both local and overall helix parameters, induced mainly by the tight interaction of Y73 and R38 in the unusually wide minor groove. Directly coupled to the widening of the groove by approximately 3A is a 21.5 degree bend of the DNA away from the bound enzyme towards the major groove, suggesting that both DNA stiffness and groove width are important in determining the sequence-dependence of the enzyme cutting rate. A second cut of the DNA which is induced by diffusion of Mn2+ into the co-crystals suggests that there are two active sites in DNase I separated by more than 15A.     

HIV requires multiple gp120 molecules for CD4-mediated infection

Binding of glycoprotein gp120 to the T cell-surface receptor CD4 is a crucial step in CD4-dependent infection of a target cell by the human immunodeficiency virus (HIV). Blocking some or all gp120 molecules on the viral surface should therefore inhibit infection. Consequently, competitive receptor inhibitors, such as soluble synthetic CD4 (sCD4), synthetic CD4 peptides and immunoglobulins, have been investigated in vitro and in vivo, but little is known about the molecular mechanisms of these inhibitors. We have now quantitatively examined blocking by soluble CD4 in the hope of gaining insight into the complex process of viral binding, adsorption and penetration. At low sCD4 concentrations, the inhibition in three HIV strains is proportional to the binding of gp120. The biological association constant (gp120-sCD4 Kassoc) for HIV-2NIHZ is (8.5 +/- 0.5) x 10(7) M-1, whereas Kassoc for HIV-1HXB3 (1.4 +/- 0.2) and HIV-1MN (1.7 +/- 0.1) x 10(9) M-1 are 15-20-fold larger. For all three viral strains, the biological Kassoc from infectivity assays is comparable to the chemical Kassoc. The inhibitory action of sCD4 at high concentrations, however, is not fully explained by simple proportionality with the binding to gp120. Positive synergy in blocking of infection occurs after about half the viral gp120s molecules are occupied, and is identical for all three viral strains, despite the large differences in Kassoc. Our method of measuring the viral-cell receptor Kassoc directly from infectivity assays is applicable to immunoglobulins, to other viruses and to assays using primary or transformed cell lines.